Anti-ngf antibodies and uses thereof

ABSTRACT

The invention provides novel anti-NGF proteins, antibodies, and NGF-binding fragments thereof which inhibit association of NGF with TrkA and/or p75 and are suitable for administration to a canine or feline subject. The invention also provides novel compositions and methods of treating pain or eliciting an analgesic effect in a canine or feline subject, comprising administering an effective amount of an anti-NGF protein, antibody or fragment thereof. The methods and compositions are used to treat or prevent NGF-related disorders.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application is a continuation of International Application No.PCT/US2022/080428 filed Nov. 23, 2022 and published as InternationalPublication No. WO 2023/097275 on Jun. 1, 2023 and which claims priorityto U.S. provisional application Ser. No. 63/282,590, filed Nov. 23,2021, and U.S. provisional application Ser. No. 63/383,173, filed Nov.10, 2022, each incorporated by reference herein in its entirety.

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which is beingsubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 22, 2022, isnamed G9432-99003.xml and is 292,984 bytes in size.

FIELD OF THE INVENTION

The invention provides novel anti-NGF proteins, antibodies, andNGF-binding fragments thereof which inhibit association of NGF with TrkAand/or p75 and are suitable for administration to a canine or felinesubject. The invention also provides novel compositions and methods oftreating pain or eliciting an analgesic effect in a canine or felinesubject, comprising administering an effective amount of an anti-NGFprotein, antibody or fragment thereof. The methods and compositions areused to treat or prevent NGF-related disorders.

BACKGROUND OF THE INVENTION

Nerve growth factor (NGF) is critical in the development and maintenanceof peripheral sympathetic and embryonic sensory neurons and of basalforebrain cholinergic neurons. NGF upregulates expression ofneuropeptides in sensory neurons and its activity is mediated throughtwo different membrane-bound receptors. Several neurotropins (NTs)including NGF bind to a low-affinity receptor identified as p75. NGFselectively binds to, and displays a high affinity for the high affinityneurotrophin receptor TrkA.

Upon neurotrophin binding, TrkA undergoes autophosphorylation as well asphosphorylates members of the MAPK pathway. The presence of this kinaseleads to cell differentiation and may play a role in specifying sensoryneuron subtypes.

NGF plays a role in several diseases and disorders, including but notlimited to pain associated with a broad range of diseases and disorders,such as pain associated with cancers, neuropathic pain, and neurogenicpain. Due to the involvement of NGF in a wide range of pain-relateddiseases and disorders, there is a need in the art for compositions andmethods useful for preventing or treating diseases and disordersassociated with NGF, particularly those associated with pain, includingin canines, felines and other animals. Particularly preferred anti-NGFcompositions are those having minimal or minimized adverse reactions,such as inflammation when administered to a subject.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The invention provides novel anti-NGF binding protein for treatment oramelioration of NGF-related disorders, particularly adapted for use indogs and cats but not limited thereby.

The invention provides binding proteins that specifically binds to NGF.In certain embodiments, the binding proteins are optimized foradministration to a canine. In certain embodiments, the binding proteinsare optimized for administration to a feline.

In an aspect, the invention provides binding proteins designed oradapted to bind NGF in the manner of an antibody, i.e. by one or morecomplementarity determining regions (CDRs). CDRs can be identified bythe international ImMunoGeneTics (IMGT) information system. Accordingly,in certain embodiments, the anti-NGF binding protein comprises anantigen binding portion that comprises one or more of (a) a heavy chaincomplementarity determining region 1 (VH-CDR1) comprising the amino acidsequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO:146), wherein X₁ comprises A, G, orN, X₂ comprises L or M, X₃ comprises A, D, E, or S, X₄ comprises F, I,L, M, or V, X₅ comprises N or T, X₆ comprises E, S, or T, X₇ comprisesG, H, N, S, or Q, and X₈ comprises A or S; (b) a heavy chaincomplementarity determining region 2 (VH-CDR2) comprising the amino acidsequence X₁X₂SNX₅GT (SEQ ID NO:147), wherein X₁ comprises I or L, X₂comprises W or Y, and X₅ comprises G or R; (c) a heavy chaincomplementarity determining region 3 (VH-CDR3) comprising the amino acidsequence AX₂IX₄X₅YX₇X₈X₉YLX₁₂X₁₃YX₁₅X₁₆X₁₇ (SEQ ID NO:148), wherein X₂comprises D, E, K, N, Q, S, or T, X₄ comprises W or Y, X₅ comprises F,H, W, or Y, X₇ comprises D or E, X₈ comprises A or S, X₉ comprises D orY, X₁₂ comprises H or Y, X₁₃ comprises F or W, X₁₅ comprises F, I, L, W,or Y, X₁₆ comprises D or Q, and X₁₇ comprises F, I, L, M, W, or Y; (d) alight chain complementarity determining region 1 (VL-CDR1) comprisingthe amino acid sequence X₁X₂IX₄X₅X₆ (SEQ ID NO:149), wherein X₁comprises D, E, or K, X₂ comprises A, G, or N, X₄ comprises G, N, Q orS, X₅ comprises N or S, X₆ comprises A, G, N, S or T; (e) a light chaincomplementarity determining region 2 (VL-CDR2) comprising the amino acidsequence AX₂X₃ (SEQ ID NO:150), wherein X₂ comprises A, S, or T, X₃comprises A, D, E, N, Q, S, or T; and (f) a light chain complementaritydetermining region 3 (VL-CDR3) comprising the amino acid sequenceQX₂GX₄X₅X₆PX₈T (SEQ ID NO:151), wherein X₂ comprises H or Q, X₄comprises F, H, W, or Y, X₅ comprises K or Q, X₆ comprises F or W, andX₈ comprises L or M.

In certain embodiments, the anti-NGF binding protein comprises anantigen binding portion which comprises (a) a heavy chaincomplementarity determining region 1 (VH-CDR1) comprising the amino acidsequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO:152), wherein X₁ comprises A or G,X₂ comprises L or M, X₃ comprises E or S, X₄ comprises F or L, X₅comprises N or T, X₆ comprises E, S, or T, X₇ comprises H, N, or S, andX₈ comprises A or S; (b) a heavy chain complementarity determiningregion 2 (VH-CDR2) comprising the amino acid sequence X₁WSNX₅GT (SEQ IDNO:153), wherein X₁ comprises I or L, X₅ comprises G or R; (c) a heavychain complementarity determining region 3 (VH-CDR3) comprising theamino acid sequence AX₂IYYYX₇ADYLHXDYX₁₅DX₁₇ (SEQ ID NO:154), wherein X₂comprises N, Q, S, or T, X₇ comprises D or E, X₁₃ comprises F or W, X₁₅comprises F, I, L, W, or Y, and X₁₇ comprises F, I, L, or M; (d) a lightchain complementarity determining region 1 (VL-CDR1) comprising theamino acid sequence X₁GIX₄X₅X₆ (SEQ ID NO:155), wherein X₁ comprises Dor E, X₄ comprises N, Q, or S, X₅ comprises N or S, X₆ comprises G, N, Sor T; (e) a light chain complementarity determining region 2 (VL-CDR2)comprising the amino acid sequence ATX₃ (SEQ ID NO:156), wherein X₃comprises D, E, N, Q, or S; and (f) a light chain complementaritydetermining region 3 (VL-CDR3) comprising the amino acid sequenceQQGX₄X₅X₆PX₈T (SEQ ID NO:157), wherein X₄ comprises F, H, W, or Y, X₅comprises K or Q, X₆ comprises F or W, and X₈ comprises L or M.

In certain embodiments, the anti-NGF binding protein comprises a heavychain CDR1 set forth in FIG. 1 . In certain embodiments, the anti-NGFbinding protein comprises a heavy chain CDR2 set forth in FIG. 1 . Incertain embodiments, the anti-NGF binding protein comprises a heavychain CDR3 set forth in FIG. 1 . In certain embodiments, the anti-NGFbinding protein comprises a light chain CDR1 set forth in FIG. 2 . Incertain embodiments, the anti-NGF binding protein comprises a lightchain CDR2 set forth in FIG. 2 . In certain embodiments, the anti-NGFbinding protein comprises a light chain CDR3 set forth in FIG. 2 .

In certain embodiments, the anti-NGF binding protein comprises heavychain CDRs of a heavy chain variable domain set forth in FIG. 1 .

In certain embodiments, the anti-NGF binding protein comprises a heavychain variable domain (V_(H)) at least 80%, at least 85%, at least 90%,at least 93%, at least 95%, at least 98%, or identical to a V_(H) domainset forth in FIG. 1 .

In certain embodiments, the anti-NGF binding protein comprises lightchain CDRs of a light chain variable domain set forth in FIG. 2 .

In certain embodiments, the anti-NGF binding protein comprises a lightchain variable domain (V_(L)) at least 80%, at least 85%, at least 90%,at least 93%, at least 95%, at least 98%, or identical to a light chainvariable domain set forth in FIG. 2 .

In certain embodiments, the anti-NGF binding protein comprises V_(H) andV_(L) from an Fv set forth in FIG. 1 and FIG. 2 .

In FIG. 1 and FIG. 2 , CDRs are identified by the IMGT system.Alternatively, CDRs can be identified according to the Kabat numberingsystem or the Chothia numbering system. Accordingly, in certainembodiments, the anti-NGF binding protein comprises an antigen bindingportion that comprises one or more of VH-CDR1, VH-CDR2, VH-CDR3,VL-CDR1, VL-CDR2, and VL-CDR3 according to the Kabat or Chothianumbering system as further set forth herein.

In paring of V_(H) and V_(L) domains described herein, any V_(H) domaincan be used with any V_(L) domain. Similarly, the CDRs of any V_(H)domain can be used with the CDRs of any V_(L) domain. In an embodiment,an antibody of the invention comprises V_(H) CDRs of SEQ ID NO:137(SC-42_101) and V_(L) CDRs of SEQ ID NO:3 (SC-42_006). In an embodimentwhich comprises the amino acid arginine at position 55 in VH-CDR2, anantibody of the invention comprises V_(H) CDRs of SEQ ID NO:207(SC-42_101R) and V_(L) CDRs of SEQ ID NO:3 (SC-42_006).

In certain embodiments, an antibody of the invention incorporate V_(H)and V_(L) domains that were selected together, i.e. identified in thesame clone. V_(H) and V_(L) clones selected together are identified ashaving the same clone name in FIG. 1 as in FIG. 2 . Similarly, the CDRsof a V_(H) domain identified by clone name in FIG. 1 can be used withthe CDRs of the V_(L) domain identified by the same clone name in FIG. 2. The paired V_(H) and V_(L) domains can further comprise conservativesubstitutions, such as but not limited to conservative variationobserved at specific positions of V_(H) CDRs and V_(L) CDRs, positionsadjacent to V_(H) and V_(L) CDRs and positions of the V_(H) domains andV_(L) domains set forth herein.

In certain embodiments, an antibody of the invention comprises V_(H) andV_(L) CDRs of clone 2166, SC-42_006, SC-42_007, SC-42_008, SC-42_010,SC-42_011, SC-42_023, SC-42_032, SC-42_045, SC-42_047, SC-42_048,SC-42_052, SC-42_070, SC-42_073, SC-42_077, SC-42_082, SC-42_090, orSC-42_101 (FIG. 1 and FIG. 2 ).

In certain embodiments, an antibody of the invention comprises V_(H) andV_(L) domains at least 80%, at least 85%, at least 90%, at least 93%, atleast 95%, at least 98%, or identical to those of clone 2166, SC-42_006,SC-42_007, SC-42_008, SC-42_010, SC-42_011, SC-42_023, SC-42_032,SC-42_045, SC-42_047, SC-42_048, SC-42_052, SC-42_070, SC-42_073,SC-42_077, SC-42_082, SC-42_090, or SC-42_101.

In certain embodiments, a felinized anti-NGF binding protein comprises(a) a heavy chain complementarity determining region 1 (VH-CDR1)comprising the amino acid sequence X₁LX₃X₄X₅X₆X₇X₈MX₁₀ (SEQ ID NO:208),wherein X₁ comprises A, G, L, N, or Q, X₃ comprises A, D, E, G, H, I, M,S, T, or Y, X₄ comprises L, M, or V, X₅ comprises A, M, N, R, S, T, orV, X₆ comprises A, E, G, H, K, R, S, or T, X₇ comprises A, D, H, I, N,Q, S, T, or Y, X₈ comprises A or S, and X₁₀ comprises S or V; (b) aheavy chain complementarity determining region 2 (VH-CDR2) comprisingthe amino acid sequence X₁X₂X₃X₄X₅GTX₈YX₁₀DX₁₂VX₁₄ (SEQ ID NO:209),wherein X₁ comprises I or L, X₂ comprises W or Y; X₃ comprises A, P, orS, X₄ comprises D, E, N, Q, R, or S, X₅ comprises G, R, or Y, X₈comprises D or Y, X₁₀ comprises D, E, H, S, or T, X₁₂ comprises D or S,and X₁₄ comprises D, E, or K; (c) a heavy chain complementaritydetermining region 3 (VH-CDR3) comprising the amino acid sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀LX₁₂X₁₃X₁₄FX₁₆X₁₇ (SEQ ID NO:210), wherein X₁comprises A, D, E, K, N, Q, S, or T, X₂ comprises A, D, E, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y, X₃ comprises I, L, W, or Y, X₄comprises F, T, W, or Y, X₅ comprises F, H, or Y, X₆ comprises H or Y,X₇ comprises D or E, X₈ comprises A, S, or V, X₉ comprises D, E, H, K,N, Q, or Y, Xu) comprises F, H, or Y, X₁₂ comprises H or Y, X₁₃comprises F or W, X₁₄ comprises D, I, L, W, or Y, X₁₆ comprises D or Q,and X₁₇ comprises E, F, H, I, L, M, N, P, W, or Y; (d) a light chaincomplementarity determining region 1 (VL-CDR1) comprising the amino acidsequence X₁ASX₄X₅X₆X₇X₈X₉LX₁₁ (SEQ ID NO:211), wherein X₁ comprises F orR, X₄ comprises E, K, or N, X₅ comprises A, or G, X₆ comprises I, L, orV, X₇ comprises A, D, G, L, P, Q, S, V, or Y, X₈ comprises K, Q, N, S,or Y, X₉ comprises A, D, E, F, G, H, K, L, N, Q, R, S or T, and X₁₁comprises A, G, or S; (e) a light chain complementarity determiningregion 2 (VL-CDR2) comprising the amino acid sequence AX₂X₃X₄X₅X₆X₇ (SEQID NO:212), wherein X₂ comprises A, D, L, Q, S, T, V, or Y, X₃ comprisesD, E, K, N, Q, or S, X₄ comprises H, I, K, L, M, N, or V; X₅ comprises Hor L, X₆ comprises H, I, L, or M, and X₇ comprises D, E, N, S, or T; and(f) a light chain complementarity determining region 3 (VL-CDR3)comprising the amino acid sequence QQX₃X₄X₅X₆X₇X₈T (SEQ ID NO:213),wherein X₃ comprises G or Y, X₄ comprises D, F, G, H, K, L, R, S, T, V,W, or Y, X₅ comprises E, K, Q, R, or S, X₆ comprises I, F, T, or W, X₇comprises E or P, and X₈ comprises L, M, or W.

In certain embodiments, the anti-NGF binding protein comprises a heavychain CDR1 set forth in FIG. 17A. In certain embodiments, the anti-NGFbinding protein comprises a heavy chain CDR2 set forth in FIG. 17A. Incertain embodiments, the anti-NGF binding protein comprises a heavychain CDR3 set forth in FIG. 17A. In certain embodiments, the anti-NGFbinding protein comprises a light chain CDR1 set forth in FIG. 17B. Incertain embodiments, the anti-NGF binding protein comprises a lightchain CDR2 set forth in FIG. 17B. In certain embodiments, the anti-NGFbinding protein comprises a light chain CDR3 set forth in FIG. 17B.

In certain embodiments, the anti-NGF binding protein comprises heavychain CDRs of a heavy chain variable domain set forth in FIG. 17A.

In certain embodiments, the anti-NGF binding protein comprises a heavychain variable domain (V_(H)) at least 80%, at least 85%, at least 90%,at least 93%, at least 95%, at least 98%, or identical to a V_(H) domainset forth in FIG. 17A.

In certain embodiments, the anti-NGF binding protein comprises lightchain CDRs of a light chain variable domain set forth in FIG. 17B.

In certain embodiments, the anti-NGF binding protein comprises a lightchain variable domain (V_(L)) at least 80%, at least 85%, at least 90%,at least 93%, at least 95%, at least 98%, or identical to a light chainvariable domain set forth in FIG. 17B.

The V_(H) CDRs set forth in FIG. 17A can be used with the V_(L) CDRs setforth in FIG. 17B in any combination. The V_(H) domains set forth inFIG. 17A can be used with the V_(L) domains set forth in FIG. 17B in anycombination. Table 11 and Table 12 provide exemplary combinations.

In certain embodiments, the anti-NGF binding protein comprises V_(H) andV_(L) from an FIT whose V_(H) is set forth in FIG. 17A and V_(L) setforth in FIG. 17B.

In FIG. 17 , CDRs are identified by the IMGT system. Alternatively, CDRscan be identified according to the Kabat numbering system or the Chothianumbering system. Accordingly, in certain embodiments, the anti-NGFbinding protein comprises an antigen binding portion that comprises oneor more of VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3according to the Kabat or Chothia or IMGT numbering system as furtherset forth herein.

In certain embodiments, the anti-NGF binding protein comprises CDRs asdescribed above, with the further limitation that each of the CDRscomprises no more than one or two amino acid differences as compared tospecific antibody heavy and light chains described herein, for example,CDRs of the heavy and light chains whose sequences are set forth in FIG.1 , FIG. 2 , FIG. 17A, and FIG. 17B, which are of similar sequence andbind to NGF with high affinity. In certain embodiments, the anti-NGFbinding protein comprise CDRs of a V_(H) and V_(L) disclosed herein.

In certain embodiments, the anti-NGF protein comprises no more than oneor two amino acid differences per CDR as compared to specific caninizedantibody heavy and light chains described herein, for example, CDRs ofthe heavy and light chains set forth in FIG. 1 and FIG. 2 which are ofsimilar sequence and bind to NGF with the highest affinity. Suchantibodies include antibodies which comprise no more than two (2)changes per VH-CDR, i.e. 2, 1 or no changes per CDR as compared to SEQID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ IDNO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ IDNO:121, SEQ ID NO:133, SEQ ID NO:137, or SEQ ID NO:141 and no more thantwo (2) changes per VL-CDR, i.e. 2, 1 or no changes per CDR as comparedto SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:62, SEQ IDNO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ IDNO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142.

In certain embodiments, the anti-NGF protein comprises no more than oneor two amino acid differences per CDR as compared to specific felinizedantibody heavy and light chains described herein, for example, CDRs ofthe heavy and light chains set forth in FIG. 17A and FIG. 17B which areof similar sequence and bind to NGF with the highest affinity. Suchantibodies include antibodies which comprise no more than two (2)changes per VH-CDR, i.e. 2, 1 or no changes per CDR as compared to SEQID NO:141, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187,SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:198, SEQ IDNO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:205, or SEQ ID NO:206and no more than two (2) changes per VL-CDR, i.e. 2, 1 or no changes perCDR as compared to SEQ ID NO:142, SEQ ID NO:191, SEQ ID NO:192, SEQ IDNO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQID NO:199, SEQ ID NO:201, or SEQ ID NO:203.

Mutations and combinations thereof, within CDRs and among CDRs,including allowed mutations and advantageous mutations are evident fromthe sequence datasets shown in FIG. 1 and FIG. 2 , and FIG. 17A and FIG.17B. For example, by comparing sequence variability or lack thereof atvarious CDR positions in the datasets as a whole, one can observe CDRlocations at which particular amino acids are beneficial for binding.Similarly, by comparing sequence variability among V_(H) chains or amongV_(L) chains from the same germline, one can observe CDR locations atwhich amino acid changes may be cooperative. The dataset further allowson to identify CDR positions that are likely to be critical for binding.

Certain antibodies disclosed herein were selected from canine or felinelibraries on the basis of CDR sequence similarity to other anti-NGFantibodies. Accordingly both CDRs and FRs are canine-like or feline-likeand there will be observed some degree of uniformity among antibodyheavy and light chains resulting from the same germline sequence. It isunderstood that such uniformity is not a necessity but a consequence ofthe caninization and felinization methods employed. It is alsounderstood that a substantial degree of sequence variability is allowedor can be introduced into FRs that is not detrimental to antigenbinding. In certain embodiments, the anti-NGF protein comprises a heavychain framework (FR1H+FR2H+FR3HH) at least 75%, or at least 80%, or atleast 85%, or at least 90%, or at least 93%, or at least 95% identicalto a heavy chain set forth in FIG. 1 . In certain embodiments, theanti-NGF protein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H)at least 75%, or at least 80%, or at least 85%, or at least 90%, or atleast 93%, or at least 95% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:31, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ IDNO:103, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQID NO:137, or SEQ ID NO:141.

In certain embodiments, the anti-NGF protein comprises a heavy chainframework (FR1H+FR2H+FR3HH) at least 75%, or at least 80%, or at least85%, or at least 90%, or at least 93%, or at least 95% identical to aheavy chain set forth in FIG. 17A. In certain embodiments, the anti-NGFprotein comprises a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%,or at least 95% identical to SEQ ID NO:141, SEQ ID NO:184, SEQ IDNO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQID NO:190, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204,SEQ ID NO:205, or SEQ ID NO:206.

In certain embodiments, the anti-NGF protein comprises a light chainframework (FR1+FR2+FR3+FR4) at least 75%, or at least 80%, or at least85%, or at least 90%, or at least 93%, or at least 95% identical to alight chain set forth in FIG. 2 . In certain embodiments, the anti-NGFprotein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%,or at least 95% identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQ IDNO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ IDNO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ ID NO:138, orSEQ ID NO:142.

In certain embodiments, the anti-NGF protein comprises a light chainframework (FR1+FR2+FR3+FR4) at least 75%, or at least 80%, or at least85%, or at least 90%, or at least 93%, or at least 95% identical to alight chain set forth in FIG. 17B. In certain embodiments, the anti-NGFprotein comprises a light chain framework (FR1L+FR2L+FR3L+FR4L) at least75%, or at least 80%, or at least 85%, or at least 90%, or at least 93%,or at least 95% identical to SEQ ID NO:142, SEQ ID NO:191, SEQ IDNO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQID NO:197, SEQ ID NO:199, SEQ ID NO:201, or SEQ ID NO:203.

For a discussion of naturally conserved networks of amino acids thatsupport antibody V(H) and V(L) function, see, e.g., Wang et al.,Conserved amino acid networks involved in antibody variable domaininteractions. Proteins 2009 July; 76(1):99-114. Wang identifiesconserved and non-conserved amino acid pairs in antibody V_(H) and V_(L)domains, the V_(H)-C_(H)1 variable-constant domain interface, as well asin camelid V_(HH) domains, which have evolved to lack interactions withV_(L) and C_(H)1. In certain embodiments, mutations are introduces tooptimize biopharmaceutical and biophysical properties, such as efficacy,safety, and manufacturability, and stability of therapeutic antibodies.See, e.g. Douillard et al., Optimization of an Antibody Light ChainFramework Enhances Expression, Biophysical Properties andPharmacokinetics. Antibodies (Basel) 2019 Sep. 6; 8(3):46.

In certain embodiments, the invention provides an isolated, recombinantNGF-binding protein wherein the variable heavy chain comprises an aminoacid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a heavychain variable domain set forth in FIG. 1 . In certain embodiments, theheavy chain variable domain comprises an amino acid sequence having atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to SEQ ID NO:13, SEQ ID NO:31, SEQ IDNO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ IDNO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, or SEQ ID NO:137.

In certain embodiments, the invention provides an isolated, recombinantNGF-binding protein wherein the variable light chain comprises an aminoacid sequence having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a lightchain variable domain set forth in FIG. 2 . In certain embodiments, thelight chain variable domain comprises an amino acid sequence having atleast about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to SEQ ID NO:14, SEQ ID NO:32, SEQ IDNO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ IDNO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, or SEQ ID NO:138.

In another aspect, the invention provides a nucleic acid that encodes ananti-NGF protein of the invention. In another aspect, the inventionprovides a vector which comprises a nucleic acid that encodes ananti-NGF protein of the invention.

In another aspect, the invention provides a cell which comprises anucleic acid of vector or the invention or expresses an anti-NGF proteinof the invention.

The anti-NGF binding proteins, such as but not limited to antibodies andantibody fragments, specifically bind NGF which inhibits the associationof NGF with TrkA and further inhibits the association of NGF with p75.In certain embodiments, these novel anti-NGF binding proteins aresuitable for detecting NGF, and for treating pain and pain-associateddisorders and conditions, e.g., pain associated with inflammation,cancer, specific pain and inflammation associated disorders, especiallypain-associated disorders associated with elevated NGF levels, and maybe administered alone or in association with another active agent, suchas but not limited to another biologic, an NSAID or opioid analgesic.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. All rights to explicitly disclaim anyembodiments that are the subject of any granted patent(s) of applicantin the lineage of this application or in any other lineage or in anyprior filed application of any third party is explicitly reserved.Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B shows an alignment of amino acid sequences of exemplary V_(H)heavy chain variable domains of the invention. The aligned variabledomains are divided in two parts: part (A) shows the N-terminal end andpart (B) shows the C-terminal end. For alignment purposes frameworks(FRs) and complementarity determining regions (CDRs) are identifiedaccording to the IMGT system. CDRs and FRs may be mapped according toother systems disclosed herein.

FIG. 2A-2B shows an alignment of amino acid sequences of exemplary V_(κ)light chain variable domains of the invention. The aligned variabledomains are divided in two parts: part (A) shows the N-terminal end andpart (B) shows the C-terminal end. For alignment purposes frameworks(FRs) and complementarity determining regions (CDRs) are identifiedaccording to the IMGT system. CDRs and FRs may be mapped according toother systems disclosed herein. Each of the V_(L) domains is suitablefor paring with any one of the V_(H) domains depicted in FIG. 1 .Together with FIG. 1 , the Clone Names indicate a selection of exemplaryVI-1-W pairs that were tested for binding.

FIG. 3 depicts inhibition of proliferation of TF-1 cells.

FIG. 4 depicts heavy chain (SEQ ID NO:144) and light chain (SEQ IDNO:145) amino acid sequences of chimeric 2166 antibody. Two residuechanges (“AA,” underlined and in bold font) were made in the Fc toeliminate effector activity. The changes are analogous to the “LALA”mutation described for human IgG1 Fc. The chimeric 2166 antibodycomprises a canine IgGB heavy chain constant region and kappa lightchain constant region.

FIG. 5 is a sensorgram for canine 2166 chimeric antibody binding tocanine NGF. The NGF concentrations were 0.78, 1.56, 3.12, 6.25, and 12.5nM.

FIG. 6 is a sensorgram for canine NGF only binding to canine p75-Fc. TheNGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM.

FIG. 7 is a sensorgram for canine NGF only binding to canine TrkA-Fc.The NGF concentrations were 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM.

FIG. 8 is a sensorgram for canine 2166 chimeric antibody-NGF mixturebinding to canine p75-Fc. The NGF concentrations were 12.5, 25, and 50nM.

FIG. 9 is a sensorgram for canine 2166 chimeric antibody-NGF mixturebinding to canine TrkA-Fc. The NGF concentrations were 12.5, 25, and 50nM.

FIG. 10 shows sensorgrams of 70 caninized clones binding to canine NGF.The NGF concentrations were 0.23, 0.69, 2.06, 6.17, and 18.52 nM

FIG. 11 is a sensorgram for canine NGF only binding to canine p75-Fc.The NGF concentrations 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM.

FIG. 12 is a sensorgram for canine NGF only binding to canine TrkA-Fc.The NGF concentrations 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 nM.

FIG. 13 is a sensorgram for caninized SC42_101 antibody-NGF mixturebinding to canine p75-Fc. The NGF concentrations were 12.5, 25, and 50nM.

FIG. 14 is a sensorgram for caninized SC42_101 antibody-NGF mixturebinding to canine TrkA-Fc. The NGF concentrations were 12.5, 25, and 50nM.

FIG. 15 depicts the V_(H) (SEQ ID NO:141) and V_(L) (SEQ ID NO:142)amino acid sequence of a felinized anti-NGF antibody.

FIG. 16 is a sensorgram for felinized clone 101 binding to NGF. The NGFconcentrations were 1.23, 3.7, 11, 33, and 100 nM.

FIG. 17A-17B shows alignments of amino acid sequences of exemplaryfelinized and affinity matured felinized V_(H) heavy chain variabledomains (A) and V_(κ) light chain variable domains (B) of the invention.For alignment purposes frameworks (FRs) and complementarity determiningregions (CDRs) are identified according to the IMGT system. CDRs and FRsmay be mapped according to other systems disclosed herein. See, e.g.,Table 4 and CDRs defined using a combination of Kabat and IMGTmethodology.

FIG. 18A-18H shows sensorgrams for affinity-matured feline antibodies:(A) clone 101; (B) AHF17602; (C)SC-184_76; (D)SC-184_76-Arg; (E)SC-102;(F)SC-184_102-Arg; (G) SC-110; (H)SC-184_110-Arg. The NGF concentrationswere 50, 25, 12.5, 6.25, 3.125, and 1.56 nM.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

DETAILED DESCRIPTION OF THE INVENTION

According to certain exemplary embodiments of the present invention, theNGF binding protein is an anti-NGF antibody or antigen-binding fragmentthereof. The term “antibody,” as used herein, includes immunoglobulinmolecules comprising four polypeptide chains, two heavy (H) chains andtwo light (L) chains inter-connected by disulfide bonds, as well asmultimers thereof (e.g., IgM). In a typical antibody, each heavy chaincomprises a heavy chain variable region (abbreviated herein as HCVR orV_(H)) and a heavy chain constant region. The heavy chain constantregion comprises three domains, C_(H)1, C_(H)2 and C_(H)3. Each lightchain comprises a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region comprises one domain (C_(L)). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDRs), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the invention,the FRs of the anti-NGF antibody (or antigen-binding portion thereof)may be identical to the canine germline sequences, or may be naturallyor artificially modified. An amino acid consensus sequence may bedefined based on a side-by-side analysis of two or more CDRs.

Antibody residues that have a substantial impact on affinity andspecificity of binding to target antigen are primarily located in CDRs.Kabat et al. compiled and aligned immunoglobulin heavy and light chainsequences and were the first to propose a standardized numbering schemefor the variable regions of immunoglobulins identifying conserved andhypervariable regions and residues. (Kabat E A et al., 1979, Sequencesof Immunoglobulin Chains: Tabulation and Analysis of Amino AcidSequences of Precursors, V-regions, C-regions, J-Chain andBP-Microglobulins, Department of Health, Education, and Welfare, PublicHealth Service, National Institutes of Health). While the Kabat systemis a widely adopted standard for numbering antibody residues, thehypervariable regions defined by Kabat do not exactly match with thestructural aspects of antigen-binding loops. Chothia and Lesk developeda structure-based numbering scheme by aligning crystal structures ofantibody variable regions and classified CDR loops in a small number of“canonical” classes (Chothia C, et al., 1987, Canonical structures forthe hypervariable regions of immunoglobulins. J. Mol. Biol. 196:901-17.doi: 10.1016/0022-2836(87)90412-8). An advantage of the Chothianumbering scheme is that topologically aligned residues from differentantibodies are localized at the same position number and the Chothia CDRdefinition corresponds in most antibody sequences to the structuralantigen-binding loop. Lefranc introduced a new system based on germ-linesequences intended to standardize numbering for all proteins of theimmunoglobulin superfamily, including T cell receptor chains.(Giudicelli V et al., 1997, IMGT, the international ImMunoGeneTicsdatabase. Nucleic Acids Res. 25:206-11), which was then extended toentire variable domains (Lefranc M-P et al., 2003, IMGT unique numberingfor immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains. Dev Comp Immunol. 27:55-77. doi:10.1016/S0145-305X(02)00039-3). Additional numbering systems have beenproposed to align unconventional frameworks (Abhinandan K R et al.,2008, Analysis and improvements to Kabat and structurally correctnumbering of antibody variable domains. Mol Immunol. 45:3832-9. doi:10.1016/j.molimm.2008.05.022) and to subdivide variable chain sequencesinto multiple fragments including structurally invariant “cores”(Gelfand et al., 1998, Algorithmic determination of core positions inthe V_(L) and V_(H) domains of immunoglobulin molecules. J Comput Biol.(1998) 5:467-77). In certain embodiments of the invention, CDR residuesare identified according to such a standard system as set forth above.In certain embodiments, antibodies of the invention are identified byall or a subset of Kabat CDR residues of the antibody sequences setforth herein. In certain embodiments, antibodies of the invention areidentified by all or a subset of Chothia CDR residues of the antibodysequences set forth herein. In certain embodiments, antibodies of theinvention are identified by all or a subset of IMGT CDR residues of theantibody sequences set forth herein. In certain embodiments, antibodiesof the invention are identified by CDR residues defined by two or moresystems, comprising e.g., but not limited to, all or a subset ofresidues of VH-CDR1 according to Kabat, all or a subset of residues ofVH-CDR2 according to Chothia, all or a subset of residues of VH-CDR3according to Kabat, all or a subset of residues of VL-CDR1 according toKabat, all or a subset of residues of VL-CDR2 according to IMGT, and allor a subset of residues of VL-CDR3 according to Chothia. Table 1 showsthe correspondence of FRs and CDRs for the antibody sequences shown inFIGS. 1 and 2 .

TABLE 1 CDR amino acids VH-FR1 VH-CDR1 VH-FR2 VH-CDR2 VH-FR3 VH-CDR3VH-FR4 IMGT 1-25 26-33 34-50 51-57 58-95 96-112 113-123 Kabat 1-30 31-3536-49 50-65 66-97 98-112 113-123 Chothia 1-25 26-32 33-51 52-56 57-9798-112 113-123 VL-FR1 VL-CDR1 VL-FR2 VL-CDR2 VL-FR3 VL-CDR3 VL-FR4 IMGT1-26 27-32 33-49 50-52 53-88 89-97  98-107 Kabat 1-23 24-34 35-49 50-5657-88 89-97  98-107 Chothia 1-25 26-32 33-49 50-52 53-90 91-96  97-107

Identifying CDRs according to Kabat, in certain embodiments, a caninizedanti-NGF binding protein comprises an antigen binding portion thatcomprises one or more of (a) a heavy chain complementarity determiningregion 1 (VH-CDR1) comprising the amino acid sequence X₁X₂X₃X₄X₅ (SEQ IDNO:158), wherein X₁ comprises E, S, or T, X₂ comprises G, H, N, S, or Q,X₃ comprises A or S; and X₄ comprises I, M, or V, and X₅ comprises D orS; (b) a heavy chain complementarity determining region 2 (VH-CDR2)comprising the amino acid sequence X₁X₂X₃SNX₆GTX₉YX₁₁X₁₂AX₁₄X₁₅X₁₆ (SEQID NO:159), wherein X₁ comprises V, L, M, or T, X₂ comprises I or L, X₃comprises W or Y, X₆ comprises G or R, X₉ comprises D, Q, or S, X₁₁comprises A, N, or T, X₁₂ comprises D or S, X₁₄ comprises I or V, X₁₅comprises E or K, and X₁₆ comprises G or S; (c) a heavy chaincomplementarity determining region 3 (VH-CDR3) comprising the amino acidsequence IX₂X₃YX₅X₆X₇YLX₁₀X₁₁YX₁₃X₁₄X₁₅, (SEQ ID NO:160), wherein X₂comprises W or Y, X₃ comprises F, H, W, or Y, X₅ comprises D or E, X₆comprises A or S, X₇ comprises D or Y, X₁₀ comprises H or Y, X₁₁comprises F or W, X₁₃ comprises F, I, L, W, or Y, X₁₄ comprises D or Q,and X₁₅ comprises F, I, L, M, W, or Y; (d) a light chain complementaritydetermining region 1 (VL-CDR1) comprising the amino acid sequenceX₁ASX₄X₅IX₇X₈X₉X₁₀X₁₁ (SEQ ID NO:161), wherein X₁ comprises L or R, X₄comprises D, E, or K, X₅ comprises A, G, or N, X₇ comprises G, N, Q orS, X₈ comprises N or S, X₉ comprises A, G, N, S or T, X₁₀ comprises L orV, and X₁₁ comprises A or N; (e) a light chain complementaritydetermining region 2 (VL-CDR2) comprising the amino acid sequenceAX₂X₃X₄X₅X₆X₇ (SEQ ID NO:162), wherein X₂ comprises A, S, or T, X₃comprises A, D, E, N, Q, S, or T, X₄ comprises A, E, K, L, N, Q, S, orT, X₅ comprises L, M, or N, X₆ comprises A or Q, and X₇ comprises G, D,R, S, or T; and (f) a light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence X₁X₂GX₄X₅X₆PX₈T (SEQ IDNO:163), wherein X₁ comprises H, M Q, or R, X₂ comprises H, N, Q, or S,X₄ comprises F, H, W, or Y, X₅ comprises K or Q, X₆ comprises F or W,and X₈ comprises L or M.

In certain embodiments, the caninized anti-NGF binding protein comprisesan antigen binding portion which comprises (a) a heavy chaincomplementarity determining region 1 (VH-CDR1) comprising the amino acidsequence X₁X₂X₃X₄S (SEQ ID NO:164), wherein X₁ comprises E, S or T, X₂comprises H or N, X₃ comprises A or S, X₄ comprises I or M, (b) a heavychain complementarity determining region 2 (VH-CDR2) comprising theamino acid sequence TIWSNX₆GTDYX₁₁X₁₂AVKG (SEQ ID NO:165), wherein X₆comprises G or R, X₁₁ comprises A or T, and X₁₂ comprises D or S; (c) aheavy chain complementarity determining region 3 (VH-CDR3) comprisingthe amino acid sequence IYYYX₅ADYLX₁₀X₁₁YX₁₃DX₁₅ (SEQ ID NO:166),wherein X₅ comprises D or E, X₁₀ comprises H or Y, X₁₁ comprises F or W,X₁₃ comprises F, I, L, W, or Y, and X₁₅ comprises F, I, L, or M; (d) alight chain complementarity determining region 1 (VL-CDR1) comprisingthe amino acid sequence RASEGIX₇X₈X₉X₁₀A (SEQ ID NO:167), wherein X₇comprises N, Q, or S, X₈ comprises N or S, X₉ comprises G, N, S or T,and X₁₀ comprises L or V; (e) a light chain complementarity determiningregion 2 (VL-CDR2) comprising the amino acid sequence ATX₃X₄LX₆X₇ (SEQID NO:168), wherein X₃ comprises A, D, E, N, Q, or S, X₄ comprises E, K,Q, or S, X₆ comprises A or Q, and X₇ comprises R or T; and (f) a lightchain complementarity determining region 3 (VL-CDR3) comprising theamino acid sequence QQGX₄X₅X₆PLT (SEQ ID NO:169), wherein X₄ comprisesF, H, W, or Y, X₅ comprises K or Q, and X₆ comprises F or W.

Identifying CDRs according to Chothia, in certain embodiments, theanti-NGF binding protein comprises an antigen binding portion thatcomprises one or more of (a) a heavy chain complementarity determiningregion 1 (VH-CDR1) comprising the amino acid sequence X₁X₂X₃X₄X₅X₆X₇(SEQ ID NO:170), wherein X₁ comprises A, G, or N, X₂ comprises L or M,X₃ comprises A, D, E, or S, X₄ comprises F, I, L, M, or V, X₅ comprisesN or T, X₆ comprises E, S, or T, and X₇ comprises G, H, N, S, or Q; (b)a heavy chain complementarity determining region 2 (VH-CDR2) comprisingthe amino acid sequence X₁SNX₄G (SEQ ID NO:171), wherein X₁ comprises Wor Y and X₄ comprises G or R; (c) a heavy chain complementaritydetermining region 3 (VH-CDR3) comprising the amino acid sequenceIX₂X₃YX₅X₆X₇YLX₁₀X₁₁YX₁₃X₁₄X₁₅, (SEQ ID NO:172), wherein X₂ comprises Wor Y, X₃ comprises F, H, W, or Y, X₅ comprises D or E, X₆ comprises A orS, X₇ comprises D or Y, X₁₀ comprises H or Y, X₁₁ comprises F or W, X₁₃comprises F, I, L, W, or Y, X₁₄ comprises D or Q, and X₁₅ comprises F,I, L, M, W, or Y; (d) a light chain complementarity determining region 1(VL-CDR1) comprising the amino acid sequence SX₂X₃IX₅X₆X₇ (SEQ IDNO:173), wherein X₂ comprises D, E, or K, X₃ comprises A, G, or N, X₅comprises G, N, Q or S, X₆ comprises N or S, X₇ comprises A, G, N, S orT; (e) a light chain complementarity determining region 2 (VL-CDR2)comprising the amino acid sequence AX₂X₃ (SEQ ID NO:174), wherein X₂comprises A, S, or T, and X₃ comprises A, D, E, N, Q, S, or T; and (f) alight chain complementarity determining region 3 (VL-CDR3) comprisingthe amino acid sequence GX₂X₃X₄PX₆ (SEQ ID NO:175), wherein X₂ comprisesF, H, W, or Y, X₃ comprises K or Q, X₄ comprises F or W, and X₆comprises L or M.

In certain embodiments, the anti-NGF binding protein comprises anantigen binding portion which comprises (a) a heavy chaincomplementarity determining region 1 (VH-CDR1) comprising the amino acidsequence X₁X₂X₃X₄X₅X₆X₇ (SEQ ID NO:176), wherein X₁ comprises A, G, orN, X₂ comprises L or M, X₃ comprises A, E, or S, X₄ comprises F or L, X₅comprises N or T, X₆ comprises E, S or T, and X₇ comprises H, N, or S;(b) a heavy chain complementarity determining region 2 (VH-CDR2)comprising the amino acid sequence WSNX₄G (SEQ ID NO:177), wherein X₄comprises G or R; (c) a heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence IYX₃YX₅ADYLX₁₀X₁₁YX₁₃DX₁₅(SEQ ID NO:178), wherein X₃ comprises F or Y, X₅ comprises D or E, X₁₀comprises H or Y, X₁₁ comprises F or W, X₁₃ comprises F, I, L, W, or Y,and X₁₅ comprises F, I, L, M, W, or Y; (d) a light chain complementaritydetermining region 1 (VL-CDR1) comprising the amino acid sequenceSX₂GIX₅X₆X₇ (SEQ ID NO:179), wherein X₂ comprises D or E, X₅ comprisesN, Q, or S, X₆ comprises N or S, and X₇ comprises G, N, S, or T; (e) alight chain complementarity determining region 2 (VL-CDR2) comprisingthe amino acid sequence ATX₃ (SEQ ID NO:180), wherein X₃ comprises D, E,N, Q, or S; and (f) a light chain complementarity determining region 3(VL-CDR3) comprising the amino acid sequence GX₂X₃X₄PX₆ (SEQ ID NO:181),wherein X₂ comprises F, H, W, or Y, X₃ comprises K or Q, X₄ comprises For W, and X₆ comprises L or M.

In another aspect, the invention provides a binding protein suitable foruse in a mammal, for example, but without limitation, a feline. Incertain embodiments, a felinized anti-NGF binding protein comprises (a)a heavy chain complementarity determining region 1 (VH-CDR1) comprisingthe amino acid sequence X₁LX₃X₄X₅X₆X₇X₈MX₁₀ (SEQ ID NO:208), wherein X₁comprises A, G, L, N, or Q, X₃ comprises A, D, E, G, H, I, M, S, T, orY, X₄ comprises L, M, or V, X₅ comprises A, M, N, R, S, T, or V, X₆comprises A, E, G, H, K, R, S, or T, X₇ comprises A, D, H, I, N, Q, S,T, or Y, X₈ comprises A or S, and X₁₀ comprises S or V; (b) a heavychain complementarity determining region 2 (VH-CDR2) comprising theamino acid sequence X₁X₂X₃X₄X₅GTX₈YX₁₀DX₁₂VX₁₄ (SEQ ID NO:209), whereinX₁ comprises I or L, X₂ comprises W or Y; X₃ comprises A, P, or S, X₄comprises D, E, N, Q, R, or S, X₅ comprises G, R, or Y, X₈ comprises Dor Y, X₁₀ comprises D, E, H, S, or T, X₁₂ comprises D or S, and X₁₄comprises D, E, or K; (c) a heavy chain complementarity determiningregion 3 (VH-CDR3) comprising the amino acid sequenceX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀LX₁₂X₁₃X₁₄FX₁₆X₁₇ (SEQ ID NO:210), wherein X₁comprises A, D, E, K, N, Q, S, or T, X₂ comprises A, D, E, G, H, I, K,L, M, N, P, Q, R, S, T, V, or Y, X₃ comprises I, L, W, or Y, X₄comprises F, T, W, or Y, X₅ comprises F, H, or Y, X₆ comprises H or Y,X₇ comprises D or E, X₈ comprises A, S, or V, X₉ comprises D, E, H, K,N, Q, or Y, X₁₀ comprises F, H, or Y, X₁₂ comprises H or Y, X₁₃comprises F or W, X₁₄ comprises D, I, L, W, or Y, X₁₆ comprises D or Q,and X₁₇ comprises E, F, H, I, L, M, N, P, W, or Y; (d) a light chaincomplementarity determining region 1 (VL-CDR1) comprising the amino acidsequence X₁ASX₄X₅X₆X₇X₈X₉LX₁₁ (SEQ ID NO:211), wherein X₁ comprises F orR, X₄ comprises E, K, or N, X₅ comprises A, or G, X₆ comprises I, L, orV, X₇ comprises A, D, G, L, P, Q, S, V, or Y, X₈ comprises K, Q, N, S,or Y, X₉ comprises A, D, E, F, G, H, K, L, N, Q, R, S or T, and X₁₁comprises A, G, or S; (e) a light chain complementarity determiningregion 2 (VL-CDR2) comprising the amino acid sequence AX₂X₃X₄X₅X₆X₇ (SEQID NO:212), wherein X₂ comprises A, D, L, Q, S, T, V, or Y, X₃ comprisesD, E, K, N, Q, or S, X₄ comprises H, I, K, L, M, N, or V; X₅ comprises Hor L, X₆ comprises H, I, L, or M, and X₇ comprises D, E, N, S, or T; (f)a light chain complementarity determining region 3 (VL-CDR3) comprisingthe amino acid sequence QQX₃X₄X₅X₆X₇X₈T (SEQ ID NO:213), wherein X₃comprises G or Y, X₄ comprises D, F, G, H, K, L, R, S, T, V, W, or Y, X₅comprises E, K, Q, R, or S, X₆ comprises I, F, T, or W, X₇ comprises Eor P, and X₈ comprises L, M, or W.

In certain embodiments, the anti-NGF binding protein comprises: VH-CDR1comprises GLSLTSX₇SMX₁₀ (SEQ ID NO:214), wherein X₇ comprises A, D, orN, and X₁₀ comprises S or V; VH-CDR2 comprises X₁X₂SNX₅GT (SEQ IDNO:215), wherein X₁ comprises I or L, X₂ comprises W or Y, and X₅comprises G or R; VH-CDR3 comprises ASIYYYX₇AX₉YLHWYFDX₁₂ (SEQ IDNO:216), wherein X₇ comprises D or E, X₉ comprises D or E, and X₁₂comprises E or F; VL-CDR1 comprises RASX₄GIX₇X₈NLS (SEQ ID NO:217),wherein X₄ comprises E or K, X₇ comprises A, Q, or S, X₈ comprises K orN; VL-CDR2 comprises AX₂X₃X₄LHS (SEQ ID NO:218), wherein X₂ comprises Qor T, X₃ comprises D or S, and X₄ comprises I, N, or V; and VL-CDR3comprises QQGX₄KWPLT (SEQ ID NO:219), wherein X₄ comprises F, W, or Y.

In certain embodiments, the anti-NGF binding protein comprises one ormore (i.e. one, two, three, four, five, or all six) CDRs of felinizedantibody 101 disclosed herein. In certain embodiments, the anti-NGFbinding protein comprises one or more (i.e. one, two, three, four, five,or all six) CDRs of an affinity matured felinized antibody disclosedherein. In certain embodiments, the anti-NGF binding protein comprisesCDRs from one or more of felinized antibody 101 and the affinity maturedvariants provided herein. In certain embodiments, the anti-NGF bindingprotein comprises V_(H) CDRs set forth in FIG. 17A. In certainembodiments, the anti-NGF binding protein comprises V_(L) CDRs set forthin FIG. 17B. In certain embodiments, the anti-NGF binding proteincomprises V_(H) CDRs of an antibody V_(H) domain set forth in FIG. 17A.In certain embodiments, the anti-NGF binding protein comprise V_(L) CDRsof an antibody V_(L) domain set forth in FIG. 17B.

According to the invention, in certain embodiments, the anti-NGF bindingprotein comprises an amino acid of a felinized antibody 101 variantdisclosed herein, for example one or more of the following amino acidsin V_(H): S28H, T30N, T30R, S31H, S35V, Y52W, S53P, G55R, G55Y, Y58D,T60D, T60E, T60H, T605, S62D, K64D, K64E, S97H, S97K, S97M, S97N, S97Q,S97T, Y99F, Y101H, D104E, D104K, D104N, D104Q, F112E, D112H, F112N,F112P; and/or in V_(L): R24F, S30A, S30L, S30P, S30Q, S30V, S30Y, N31Q,S34A, S34G, N53H, N53I, N53K, N53L, N53M, N53V, L54H, H55I, H55L, H55M,S56D, S56E, S56N, S56T. In certain embodiments, the anti-NGF bindingprotein does not comprise one or more of the above-listed amino acidvariants. For example, in certain embodiments, the anti-NGF bindingprotein does not comprise arginine of G55R. Amino acid positions areindicated by residue and number in felinized antibody 101, e.g., S28Hindicates H at the position corresponding to S28 of V_(H) or antibody101. The aforementioned positions include CDR and framework amino acidresidues.

In certain embodiments, the anti-NGF binding protein comprises one ormore of the following amino acids in V_(H): S35V, G55R, S97Q, F112E;and/or in V_(L): S30A, S30Q, N53I, N53V. Pairings of V_(H) and V_(L)chains comprising the above-described sequence variation demonstratecompatibility of the V_(H) and V_(L) mutations and interchangeability ofthe V_(H) and V_(L) domains comprising the mutations.

In certain embodiments, the binding proteins comprise a canine or acaninized antibody. In certain embodiments, the binding proteinscomprise a feline or a felinized antibody.

In certain embodiments, an amino acid residue is mutated into one thatallows the properties of the amino acid side-chain to be conserved.Examples of the properties of amino acid side chains comprise:hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic aminoacids (R, D, N, C, E, Q, G, H, K, S, T), and amino acids comprising thefollowing side chains: aliphatic side-chains (G, A, V, L, I, P);hydroxyl group-containing side-chains (S, T, Y); sulfur atom-containingside-chains (C, M); carboxylic acid- and amide-containing side-chains(D, N, E, Q); base-containing side-chains (R, K, H); andaromatic-containing side-chains (H, F, Y, W). The letters withinparenthesis indicate the one-letter amino acid codes. Amino acidsubstitutions within each group are called conservative substitutions.It is well known that a polypeptide comprising a modified amino acidsequence in which one or more amino acid residues is deleted, added,and/or substituted can retain the original biological activity (Mark D.F. et al., Proc. Natl. Acad. Sci. U.S.A. 81:5662-5666 (1984); Zoller M.J. and Smith M., Nucleic Acids Res. 10: 6487-6500 (1982); Wang A. etal., Science 224: 1431-1433; Dalbadie-McFarland G. et al., Proc. Natl.Acad. Sci. U.S.A. 79: 6409-6413 (1982)). The number of mutated aminoacids is not limited, but in general, the number falls within 40% ofamino acids of each CDR, and preferably within 35%, and still morepreferably within 30% (e.g., within 25%). The identity of amino acidsequences can be determined as described herein.

The invention provides recombinant antibodies designed or modified tominimize antigenicity in canines and felines. In certain embodiments,the antibodies are further modified to remove T cell epitopes.

As used herein, the term “canine” includes all domestic dogs, Canislupus familiaris or Canis familiaris, unless otherwise indicated.

As used herein, the term “feline” refers to any member of the Felidaefamily. Domestic cats, pure-bred and/or mongrel companion cats, and wildor feral cats are all felines.

As used herein the term “canine framework” or “feline framework” refersto the amino acid sequence of the heavy chain and light chain of acanine antibody other than the hypervariable region residues definedherein as CDR residues. With regard to a caninized antibody, in certainembodiments, canine CDRs are identified in canine antibody heavy andlight chains variable domain sequences that closely match CDRs ofNGF-binding antibodies originating in other species. In certainembodiments, native canine CDRs are replaced with the correspondingforeign CDRs (e.g., those from a rat or a mouse antibody) in bothchains. With regard to a felinized antibody, in certain embodiments,feline CDRs are identified in feline antibody heavy and light chainsvariable domain sequences that closely match CDRs of NGF-bindingantibodies originating in other species. In certain embodiments, nativefeline CDRs are replaced with the corresponding foreign CDRs (e.g.,those from a rat or a mouse antibody) in both chains. Optionally theheavy and/or light chains of the caninized or felinized antibody maycontain some mutated or foreign non-CDR residues, e.g., framework aminoacid residues that vary among germline antibody sequence or mutationsthat preserve the conformation of the foreign CDRs within the antibody.

Five major isotypes (IgA, IgG, IgM, IgD, IgE) and two forms of lightchain (κ and λ) are present in dogs. In the dog, there are four subtypesfor IgG, which are IgGA, IgGB, IgGC, and IgGD (Bergeron et al, 2014,Comparative functional characterization of canine IgG subclasses.Veterinary Immunology and Immunopathology. 157:31-41). For the cat,there are three subtypes of IgG which are IgG1a, IgG1b, and IgG2(Streitzel et al. 2014, In vitro functional characterization of felineIgGs. Vet Immunol Immunopathol 158, 214-223,doi.org/10.1016/j.vetimm.2014.01.012).

The invention provides caninized and felinized antibodies engineered tomodulate one or more effector functions or circulation half-life. Hingeand constant domains of an antibody engage host receptors or complementprotein to mediate effector functions and regulate antibody circulation.In certain embodiments, one or more effector functions is enhanced. Incertain embodiments, one or more effector functions is reduced oreliminated. In certain embodiments, antibodies of the invention comprisemodifications to modulate antibody-dependent cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC). A non-limiting example involvesengineering of canine IgGB constant region residues Met242 and/or Leu243to reduce effector function. In certain embodiments, a IgGB constantregion of the invention comprises M242A and L243A substitution. Incertain embodiments, the second constant domain (CH2) and/or the thirdconstant domain (CH3) comprises mutations and combinations of mutationsfrom wild-type designed to modulate binding to FcRn (neonatal Fc)receptor. In canine constant regions, such mutations include, withoutlimitation substitutions of Ala426, for example A426Y or A426H,substitutions of Thr286, for example T286L or T286Y, substitutions ofTyr436, for example Y436H, and combinations of such mutations includingbut not limited to A426Y+T286L, A426Y+Y436H, A426H+T286L, andA426H+T286Y. In certain embodiments a chimeric or caninized antibody ofthe invention comprises a substitution at amino acid Asn434, such as butnot limited to N434H. In feline constant regions, such mutationsinclude, without limitation substitutions of Ser428, including but notlimited to S428Y or S428L, substitutions of Gln311, including but notlimited to Q311V, substitutions of Leu309, including but not limited toL309V, substitutions of Thr286, including but not limited to T286E,substitutions of Glu380, including but not limited to E380T, andcombinations of such mutations including but not limited to S428Y+Q311V,S428Y+L309V, S428Y+Q311V+T286E, S428Y+Q311V+E380T, andS428Y+L309V+E380T. In certain embodiments a chimeric or felinizedantibody of the invention comprises a substitution at amino acid Ser428and/or Ser434 including but not limited to S428L and/or S434H.

The term “antibody,” as used herein, includes antigen-binding fragmentsof full antibody molecules. The terms “antigen-binding portion” of anantibody, “antigen-binding fragment” of an antibody, and the like, asused herein, include any naturally occurring, enzymatically obtainable,synthetic, or genetically engineered polypeptide or glycoprotein thatspecifically binds an antigen to form a complex. As used herein, theterm “specifically binds” or “binds specifically” means that an NGFbinding protein of the invention reacts or associates more frequently,more rapidly, with greater duration and/or with greater affinity withNGF than it does with alternative antigens. For example, NGF bindingprotein binds to NGF with materially greater affinity (e.g., at least2-fold or 5-fold or 10-fold or 20-fold or 50-fold or 100-fold or500-fold or 1000-fold or 10,000-fold or greater) than it does to otherproteins or peptides. In certain embodiments, the NGF-binding proteinsbinds to NGF with an equilibrium dissociation constant K_(D) for theepitope or target to which it binds of, e.g., 10⁻⁴ M or smaller, e.g.,10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M. Itwill be recognized by one of skill that an antibody that specificallybinds to a target (e.g., NGF) from one species may also specificallybind to orthologs of NGF.

Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, e.g., commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

Non-limiting examples of antigen-binding fragments include: (i) Fabfragments; (ii) F(ab′)2 fragments; (iii) Fd fragments; (iv) Fvfragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and(vii) minimal recognition units consisting of the amino acid residuesthat mimic the hypervariable region of an antibody (e.g., an isolatedcomplementarity determining region (CDR) such as a CDR3 peptide), or aconstrained FR3-CDR3-FR4 peptide. Other engineered molecules, such asdomain-specific antibodies, single domain antibodies, domain-deletedantibodies, chimeric antibodies, CDR-grafted antibodies, diabodies,triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalentnanobodies, bivalent nanobodies, etc.), small modularimmunopharmaceuticals (SMIPs), and shark variable IgNAR domains, arealso encompassed within the expression “antigen-binding fragment,” asused herein.

In certain embodiments, an antigen-binding fragment of an antibodycomprises at least one variable domain. The variable domain may be ofany size or amino acid composition and will generally comprise at leastone CDR which is adjacent to or in frame with one or more frameworksequences. In antigen-binding fragments having a V_(H) domain associatedwith a V_(L) domain, the V_(H) and V_(L) domains may be situatedrelative to one another in any suitable arrangement. For example, thevariable region may be dimeric and contain V_(H)-V_(H), V_(H)-V_(L) orV_(L)-V_(L) dimers. Alternatively, the antigen-binding fragment of anantibody may contain a monomeric V_(H) or V_(L) domain.

In certain embodiments, an antigen-binding fragment of an antibody maycontain at least one variable domain covalently linked to at least oneconstant domain. Non-limiting, exemplary configurations of variable andconstant domains that may be found within an antigen-binding fragment ofan antibody of the present invention include: (i) V_(H)-C_(H)1; (ii)V_(H)-C_(H)2; (iii) V_(H)-C_(H)3; (iv) V_(H)-C_(H)1-C_(H)2; (V)V_(H)-C_(H)1-C_(H)2-C_(H)3; (vi) V_(H)-C_(H)2-C_(H)3; (vii) V_(H)-CL;(viii) V_(L)-C_(H)1; (ix) V_(L)-C_(H)2, (x) V_(L)-C_(H)3; (xi)V_(L)-C_(H)1-C_(H)2; (xii) V_(L)-C_(H)1-C_(H)2-C_(H)3; (xiii)V_(L)-C_(H)2-C_(H)3; and (xiv) V_(L)-C_(L). In any configuration ofvariable and constant domains, including any of the exemplaryconfigurations listed above, the variable and constant domains may beeither directly linked to one another or may be linked by a full orpartial hinge or linker region. A hinge region may consist of at least 2(e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in aflexible or semi-flexible linkage between adjacent variable and/orconstant domains in a single polypeptide molecule. Moreover, anantigen-binding fragment of an antibody of the present invention maycomprise a homo-dimer or hetero-dimer (or other multimer) of any of thevariable and constant domain configurations listed above in non-covalentassociation with one another and/or with one or more monomeric V_(H) orV_(L) domain (e.g., by disulfide bond(s)).

The term “diabody (Db)” refers to a bivalent antibody fragmentconstructed by gene fusion (for example, P. Holliger et al., Proc. Natl.Acad. Sci. USA 90: 6444-6448 (1993), EP 404,097, WO 93/11161). Ingeneral, a diabody is a dimer of two polypeptide chains. In the each ofthe polypeptide chains, a light chain variable region (V_(L)) and aheavy chain variable region (V_(H)) in an identical chain are connectedvia a short linker, for example, a linker of about five residues, sothat they cannot bind together. Because the linker between the two istoo short, the V_(L) and V_(H) in the same polypeptide chain cannot forma single chain V region fragment, but instead form a dimer. Thus, adiabody has two antigen-binding domains. When the V_(L) and V_(H)regions against the two types of antigens (a and b) are combined to formV_(La)-V_(Hb) and V_(Lb)-V_(Ha) via a linker of about five residues, andthen co-expressed, they are secreted as bispecific Dbs. The antibodiesof the present invention may be such Dbs.

A single-chain antibody (also referred to as “scFv”) can be prepared bylinking a heavy chain V region and a light chain V region of an antibody(for a review of scFv see Pluckthun “The Pharmacology of MonoclonalAntibodies” Vol. 113, eds. Rosenburg and Moore, Springer Verlag, N.Y.,pp. 269-315 (1994)). Methods for preparing single-chain antibodies areknown in the art (see, for example, U.S. Pat. Nos. 4,946,778; 5,260,203;5,091,513; and 5,455,030). In such scFvs, the heavy chain V region andthe light chain V region are linked together via a linker, preferably, apolypeptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A,1988, 85, 5879-5883). The heavy chain V region and the light chain Vregion in a scFv may be derived from the same antibody, or fromdifferent antibodies. The peptide linker used to ligate the V regionsmay be any single-chain peptide consisting of 12 to 19 residues. A DNAencoding a scFv can be amplified by PCR using, as a template, either theentire DNA, or a partial DNA encoding a desired amino acid sequence,selected from a DNA encoding the heavy chain or the V region of theheavy chain of the above antibody, and a DNA encoding the light chain orthe V region of the light chain of the above antibody; and using aprimer pair that defines the two ends. Further amplification can besubsequently conducted using a combination of the DNA encoding thepeptide linker portion, and the primer pair that defines both ends ofthe DNA to be ligated to the heavy and light chain respectively. Afterconstructing DNAs encoding scFvs, conventional methods can be used toobtain expression vectors comprising these DNAs, and hosts transformedby these expression vectors. Furthermore, scFvs can be obtainedaccording to conventional methods using the resulting hosts. Theseantibody fragments can be produced in hosts by obtaining genes thatencode the antibody fragments and expressing these as outlined above.Antibodies bound to various types of molecules, such as polyethyleneglycols (PEGs), may be used as modified antibodies. Methods formodifying antibodies are already established in the art. The term“antibody” in the present invention also encompasses the above-describedantibodies.

The term “Kd” as used herein, refers to the dissociation constant of anantibody-antigen interaction. The dissociation constant, Kd, and theassociation constant, Ka, are quantitative measures of affinity. Atequilibrium, free antigen (Ag) and free antibody (Ab) are in equilibriumwith antigen-antibody complex (Ag-Ab), and the rate constants, ka andkd, quantitate the rates of the individual reactions. At equilibrium, ka[Ab][Ag]=kd [Ag-Ab]. The dissociation constant, Kd, is given by:Kd=kd/ka=[Ag][Ab]/[Ag-Ab]. Kd has units of concentration, most typicallyM, mM, nM, pM, etc. When comparing antibody affinities expressed as Kd,having greater affinity for NGF is indicated by a lower value. Theassociation constant, Ka, is given by: Ka=ka/kd=[Ag-Ab]/[Ag][Ab]. Ka hasunits of inverse concentration, most typically M⁻¹, mM⁻, nM⁻¹, pM⁻¹,etc. As used herein, the term “avidity” refers to the strength of theantigen-antibody binding taking valency into account.

The antibodies obtained can be purified to homogeneity. The antibodiescan be isolated and purified by a method routinely used to isolate andpurify proteins. The antibodies can be isolated and purified by thecombined use of one or more methods appropriately selected from columnchromatography, filtration, ultrafiltration, salting out, dialysis,preparative polyacrylamide gel electrophoresis, and isoelectro-focusing,for example (Strategies for Protein Purification and Characterization: ALaboratory Course Manual, Daniel R. Marshak et al. eds., Cold SpringHarbor Laboratory Press (1996); Antibodies: A Laboratory Manual. EdHarlow and David Lane, Cold Spring Harbor Laboratory, 1988). Suchmethods are not limited to those listed above. Chromatographic methodsinclude affinity chromatography, ion exchange chromatography,hydrophobic chromatography, gel filtration, reverse-phasechromatography, and adsorption chromatography. These chromatographicmethods can be practiced using liquid phase chromatography, such as HPLCand FPLC. Columns to be used in affinity chromatography include proteinA columns and protein G columns. For example, protein A columns includeHyper D, POROS, and Sepharose F. F. (Pharmacia). Antibodies can also bepurified by utilizing antigen binding, using carriers on which antigenshave been immobilized.

As used herein, the term “therapeutic agent” refers to any agent ormaterial that has a beneficial effect on the mammalian recipient. Thus,“therapeutic agent” embraces both therapeutic and prophylactic moleculeshaving nucleic acid or protein components.

“Treating” as used herein refers to ameliorating at least one symptomof, curing and/or preventing the development of a given disease orcondition.

The anti-NGF proteins described herein, including antibodies orfragments thereof, are useful for ameliorating, or reducing the symptomsof, or treating, or preventing, diseases and disorders associated withNGF. The anti-NGF proteins or fragments, as well as combinations withother agent, are to be administered in a therapeutically effectiveamount to subjects in need of treatment of diseases and disordersassociated with NGF in the form of a pharmaceutical composition asdescribed herein

In certain embodiments the method comprises ameliorating, or reducingthe symptoms of, or treating, or preventing pain in a subject. Incertain embodiments, the anti-NGF proteins, antibodies, or fragmentsthereof inhibit the association of NGF with TrkA and/or p75, for exampleadministered alone or in conjunction with a second agent and are used totreat, ameliorate, reduce the symptoms of, or prevent inflammatory pain,post-operative incision pain, complex, cancer pain (including but notlimited to primary or metastatic bone cancer pain), fracture pain,osteoporotic fracture pain, pain from osteoporosis, pain resulting fromburn, and other nociceptic pain.

In certain embodiments the antibody compositions and methods are usedfor ameliorating, or reducing the symptoms of, or treating, orpreventing pain of osteoarthritis (OA). OA is a slowly progressivedegenerative joint disease characterized by whole-joint structuralchanges including articular cartilage, synovium, subchondral bone andperiarticular components, leading to pain and loss of joint function.Chronic pain and OA are common in dogs and cats. 20-30% of dogs areaffected clinically and have signs of OA. Up to 40% of all cats beingaffected clinically, with 90% of all cats over 12 years of age havesigns of OA.

In dogs the most common site of OA is the hip, followed by stifle(knee), shoulder and carpus. In cats hip, stifle, carpus or spine aremost commonly affected.

The anti-NGF proteins, antibodies or antibody fragments, are optionallyadministered in combination with one or more active agents includingother analgesic agents. Such active agents include analgesic,anti-histamine, antipyretic, anti-inflammatory, antibiotic, antiviral,and anti-cytokine agents. Active agents include agonists, antagonists,and modulators of TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18,IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, HepatocyteGrowth Factor (HGF), Hepcidin, including antibodies reactive against anyof the foregoing, and antibodies reactive against any of theirreceptors. Active agents also include, without limitation,2-arylpropionic acids, aceclofenac, acemetacin, acetylsalicylic acid(Aspirin), alclofenac, alminoprofen, amoxiprin, ampyrone, arylalkanoicacids, azapropazone, benorylate/benorilate, benoxaprofen, bromfenac,carprofen, celecoxib, choline magnesium salicylate, clofezone, COX-2inhibitors, dexibuprofen, dexketoprofen, diclofenac, diflunisal,droxicam, ethenzamide, etodolac, etoricoxib, faislamine, fenamic acids,fenbufen, fenoprofen, flufenamic acid, flunoxaprofen, flurbiprofen,ibuprofen, ibuproxam, indometacin, indoprofen, kebuzone, ketoprofen,ketorolac, lomoxicam, loxoprofen, lumiracoxib, magnesium salicylate,meclofenamic acid, mefenamic acid, meloxicam, metamizole, methylsalicylate, mofebutazone, nabumetone, naproxen, n-arylanthranilic acids,nerve growth factor (NGF), oxametacin, oxaprozin, oxicams,oxyphenbutazone, parecoxib, phenazone, phenylbutazone, phenylbutazone,piroxicam, pirprofen, profens, proglumetacin, pyrazolidine derivatives,rofecoxib, salicyl salicylate, salicylamide, salicylates,sulfinpyrazone, sulindac, suprofen, tenoxicam, tiaprofenic acid,tolfenamic acid, tolmetin, and valdecoxib.

An anti-histamine can be any compound that opposes the action ofhistamine or its release from cells (e.g., mast cells). Anti-histaminesinclude but are not limited to acrivastine, astemizole, azatadine,azelastine, betatastine, brompheniramine, buclizine, cetirizine,cetirizine analogues, chlorpheniramine, clemastine, CS 560,cyproheptadine, desloratadine, dexchlorpheniramine, ebastine,epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine,loratidine, methscopolamine, mizolastine, norastemizole, phenindamine,promethazine, pyrilamine, terfenadine, and tranilast.

Antibiotics include but are not limited to amikacin, aminoglycosides,amoxicillin, ampicillin, ansamycins, arsphenamine, azithromycin,azlocillin, aztreonam, bacitracin, carbacephem, carbapenems,carbenicillin, cefaclor, cefadroxil, cefalexin, cefalothin, cefalotin,cefamandole, cefazolin, cefdinir, cefditoren, cefepime, cefixime,cefoperazone, cefotaxime, cefoxitin, cefpodoxime, cefprozil,ceftazidime, ceftibuten, ceftizoxime, ceftobiprole, ceftriaxone,cefuroxime, cephalosporins, chloramphenicol, cilastatin, ciprofloxacin,clarithromycin, clindamycin, cloxacillin, colistin, co-trimoxazole,dalfopristin, demeclocycline, dicloxacillin, dirithromycin, doripenem,doxycycline, enoxacin, ertapenem, erythromycin, ethambutol,flucloxacillin, fosfomycin, furazolidone, fusidic acid, gatifloxacin,geldanamycin, gentamicin, glycopeptides, herbimycin, imipenem,isoniazid, kanamycin, levofloxacin, lincomycin, linezolid, lomefloxacin,loracarbef, macrolides, mafenide, meropenem, meticillin, metronidazole,mezlocillin, minocycline, monobactams, moxifloxacin, mupirocin,nafcillin, neomycin, netilmicin, nitrofurantoin, norfloxacin, ofloxacin,oxacillin, oxytetracycline, paromomycin, penicillin, penicillins,piperacillin, platensimycin, polymyxin B, polypeptides, prontosil,pyrazinamide, quinolones, quinupristin, rifampicin, rifampin,roxithromycin, spectinomycin, streptomycin, sulfacetamide,sulfamethizole, sulfanilimide, sulfasalazine, sulfisoxazole,sulfonamides, teicoplanin, telithromycin, tetracycline, tetracyclines,ticarcillin, tinidazole, tobramycin, trimethoprim,trimethoprim-sulfamethoxazole, troleandomycin, trovafloxacin, andvancomycin.

Active agents also include aldosterone, beclometasone, betamethasone,corticosteroids, cortisol, cortisone acetate, deoxycorticosteroneacetate, dexamethasone, fludrocortisone acetate, glucocorticoids,hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids,and triamcinolone. Any suitable combination of these active agents isalso contemplated.

The most common form of current treatment for OA and pain related to OAis NSAIDs (which are also anti-pain medications). NSAIDs are not alwayssufficiently effective, typically need to be administered daily and noneare approved for long-term use in cats in the US. Additionally, thereare safety and tolerability concerns with the use of NSAIDS in both dogsand cats, especially with long-term treatment. NSAIDs are notrecommended to be co-administered with anti-NGF mAbs for long periods.

In certain embodiments, treatment comprises coadministration of dietarysupplements containing Omega-3 fatty acids, microlactin, and/orglucosamine/chondroitin as an aid to joint health. Adequan (polysulfatedglycosaminoglycan) is an FDA-approved disease modifying drug thatinhibits cartilage loss and may also be co-administered.

Formulations and Methods of Administration

For in vivo use, a therapeutic agent as described herein is generallyincorporated into a pharmaceutical composition prior to administration.Within such compositions, one or more therapeutic compounds as describedherein are present as active ingredient(s) (i.e., are present at levelssufficient to provide a statistically significant effect on the symptomsof cystic fibrosis, as measured using a representative assay). Apharmaceutical composition comprises one or more such compounds incombination with any pharmaceutically acceptable carrier(s) known tothose skilled in the art to be suitable for the particular mode ofadministration. In addition, other pharmaceutically active ingredients(including other therapeutic agents) may, but need not, be presentwithin the composition.

The antibodies of the present invention can be formulated according tostandard methods (see, for example, Remington's Pharmaceutical Science,latest edition, Mark Publishing Company, Easton, U.S.A), and maycomprise pharmaceutically acceptable carriers and/or additives. Thepresent invention relates to compositions (including reagents andpharmaceuticals) comprising the antibodies of the invention, andpharmaceutically acceptable carriers and/or additives. Exemplarycarriers include surfactants (for example, PEG and Tween), excipients,antioxidants (for example, ascorbic acid), coloring agents, flavoringagents, preservatives, stabilizers, buffering agents (for example,phosphoric acid, citric acid, and other organic acids), chelating agents(for example, EDTA), suspending agents, isotonizing agents, binders,disintegrators, lubricants, fluidity promoters, and corrigents. However,the carriers that may be employed in the present invention are notlimited to this list. In fact, other commonly used carriers can beappropriately employed: light anhydrous silicic acid, lactose,crystalline cellulose, mannitol, starch, carmelose calcium, carmelosesodium, hydroxypropylcellulose, hydroxypropylmethyl cellulose,polyvinylacetaldiethylaminoacetate, polyvinylpyrrolidone, gelatin,medium chain fatty acid triglyceride, polyoxyethylene hydrogenatedcastor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganicsalt, and so on. The composition may also comprise otherlow-molecular-weight polypeptides, proteins such as serum albumin,gelatin, and immunoglobulin, and amino acids such as glycine, glutamine,asparagine, arginine, and lysine. When the composition is prepared as anaqueous solution for injection, it can comprise an isotonic solutioncomprising, for example, physiological saline, dextrose, and otheradjuvants, including, for example, D-sorbitol, D-mannose, D-mannitol,and sodium chloride, which can also contain an appropriate solubilizingagent, for example, alcohol (for example, ethanol), polyalcohol (forexample, propylene glycol and PEG), and non-ionic detergent (polysorbate80 and HCO-50).

If necessary, antibodies of the present invention may be encapsulated inmicrocapsules (microcapsules made of hydroxycellulose, gelatin,polymethylmethacrylate, and the like), and made into components ofcolloidal drug delivery systems (liposomes, albumin microspheres,microemulsions, nano-particles, and nano-capsules) (for example, see“Remington's Pharmaceutical Science 16th edition”, Oslo Ed. (1980)).Moreover, methods for making sustained-release drugs are known, andthese can be applied for the antibodies of the present invention (Langeret al., J. Biomed. Mater. Res. 15: 167-277 (1981); Langer, Chem. Tech.12: 98-105 (1982); U.S. Pat. No. 3,773,919; EP Patent Application No.58,481; Sidman et al., Biopolymers 22: 547-556 (1983); EP: 133,988).

A preferred route of administration in both canines and felines is bysubcutaneous injection usually into the skin at the base of the neck. Incertain embodiments, the anti-NGF protein is packaged in an integrateddelivery system such as a pen or prefilled syringe for subcutaneousadministration. Ghil et al. describes administration of the adalimumabbiosimilar, SB5, via prefilled syringe (PFS) and autoinjector (AI) penbased on injection site pain, patient preference, and safety inrheumatoid arthritis (RA) (See Ghil et al., Usability and safety of SB5(an adalimumab biosimilar) prefilled syringe and autoinjector inpatients with rheumatoid arthritis. Curr Med Res Opin 2019 March;35(3):497-502.) Compositions of the invention are similarly administeredto canines, felines, and other mammals.

The term “therapeutically effective amount,” in reference to treating adisease state/condition, refers to an amount of a compound either aloneor as contained in a pharmaceutical composition that is capable ofhaving any detectable, positive effect on any symptom, aspect, orcharacteristics of a disease state/condition when administered as asingle dose or in multiple doses. Such effect need not be absolute to bebeneficial.

The terms “treat,” “treating” and “treatment” as used herein includeadministering a compound prior to the onset of clinical symptoms of adisease state/condition so as to prevent any symptom, as well asadministering a compound after the onset of clinical symptoms of adisease state/condition so as to reduce or eliminate any symptom, aspector characteristic of the disease state/condition. Such treating need notbe absolute to be useful.

In certain embodiments, the present therapeutic agent may besystemically administered, e.g., orally, in combination with apharmaceutically acceptable vehicle such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets, or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the active compound may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of a given unit dosage form. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. In certain embodiments, a useful dose is from about 0.1mg/kg to about 5 mg/kg or from about 0.5 mg/kg to about 2 mg/kg. Methodsfor the extrapolation of effective dosages in humans and animals ofdifferent sizes are known to the art; for example, see U.S. Pat. No.4,938,949.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

In general, however, a suitable dose will be in the range of from about0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 6 to 90 mg/kg/day, mostpreferably in the range of 15 to 60 mg/kg/day.

The compound is conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 This may be achieved, for example, by the intravenous injection of a0.05 to 5% solution of the active ingredient, optionally in saline, ororally administered as a bolus containing about 1-100 mg of the activeingredient. Desirable blood levels may be maintained by continuousinfusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusionscontaining about 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined in the appended claims.

The present invention will be further illustrated in the followingExamples which are given for illustration purposes only and are notintended to limit the invention in any way.

EXAMPLES Example 1

Generation and Characterization of Rat Antibody 2166 that Binds toCanine NGF.

Lewis rats were immunized with human NGF (R&D Systems, 256-GF-100/CF) ona weekly basis for eight weeks. The titers were measured in a flowcytometry assay using human NGF-coated beads. Beads were conjugated withhuman NGF (R&D Systems, 256-GF-100/CF) and incubated with differentdilutions of serum (1:100, 1:500, 1:2500) for 30 minutes. Beads werewashed and binding was detected by using a fluorescently labeledanti-rat IgG secondary antibody. Fluorescence was measured using theIntellicyt iQue Screener Plus. Titers were measured at a 1:2500 dilutionfor all three rats and they were ˜100-fold greater than the values ofnormal Lewis rat serum.

Lymph nodes (brachial, axillary, inguinal, popliteal and sciatic) andbone marrow from femur, tibia and pelvis were collected from rats withsignificant NGF titers. Cells from both tissues were isolated andenriched for plasma cells using flow cytometry. Enriched plasma cellsuspension was injected into AbCellera's microfluidic screening deviceswith either 91,000 or 153,000 individual nanoliter-volume reactionchambers. Single cells secreting NGF-specific antibodies were identifiedand isolated using a bead-based assay. Beads coated with anti-rat IgGantibody were flowed onto microfluidic screening devices and incubatedwith single antibody-secreting cells. The IgG secreted by plasma cellswere captured on beads using the constant region. Binding to secretedIgG immobilized onto beads was subsequently assessed using fluorescentlylabeled human NGF antigen. Positive hits were identified using machinevision and recovered using automated robotics-based protocols.Approximately 269,000 individual B cells were screened in the NGFbinding assay and 592 cells expressed antibodies recognized NGF. Fromthese positive cells, 190 unique antibody sequences were identified.Eighty-eight antibodies were selected from the 190 antibodies based onthe diversity of the clonotypes.

Single cell polymerase chain reaction and custom molecular biologyprotocols generated NGS sequencing libraries (MiSeq, Illumina) usingautomated workstations (Bravo, Agilent). Sequencing data were analyzedusing a custom bioinformatics pipeline to yield paired heavy and lightchain sequences for each recovered antibody-secreting cell (Jones etal., 2020, bioRxiv 2020.09.30.318972. doi: 10.1101/2020.09.30.318972).The amino acid sequences of the heavy and light variable domains of ratantibody 2166 are shown in FIG. 1 and FIG. 2 respectively and the CDRsare indicated. The variable (V(D)J) region of each antibody chain wassynthesized and inserted into mammalian expression plasmids using acustom, automated high-throughput cloning pipeline.

The expression vectors were transfected into Expi293-F cells (Gibco,ThermoFisher Scientific) in 24 deep well plates using the manufacturer'srecommended protocol. Four days post-transfection, the conditionedmedium was purified with protein A beads and the antibody was eluted bythe addition of 100 mM glycine, pH 2.0 and neutralized to pH 7.0 by theaddition of 1 M Tris-HCL, pH 8.0. The neutralized antibodies were bufferexchanged into PBS, pH 7.2.

The analytics for the purified antibodies included CE-SDS (denaturingcapillary sodium dodecyl sulfate gel electrophoresis) and DSF(differential scanning fluorimetry). The CE-SDS was used to determinethe purity of the purified antibodies and was completed by using theLabChip GXII Touch instrument (Perkin Elmer). Two microliters ofantibody solution at a concentration of 350 μg/mL in PBS was mixed witha non-reducing denaturing buffer solution (Perkin Elmer) and incubatedat 70° C. for 10 minutes. Separation and detection were performed usingthe HT Antibody Analysis 200 assay setting on the LabChip instrument(Perkin Elmer). The fluorescence data was analyzed using the LabChip GXReviewer Software (Perkin Elmer), with percent purity. The percentpurity of the rat monoclonal antibody 2166 was 96%.

The melting point (Tm) of antibodies was assessed by differentialscanning fluorimetry (DSF) using the SYPRO™ Orange fluorescence probe(5000× concentrated solution, Thermo Fisher Scientific). 6 μL of mAbsolution at 350 μg/mL in PBS was mixed with 6 μL of a 19× concentratedSYPRO™ Orange solution diluted in PBS. Thermal unfolding as assessed bya change in fluorescence was measured on a Bio-Rad C1000 Touch ThermalCycler instrument (Bio-Rad Laboratories) using a CFX96 Real-Time Systemreader head (Bio-Rad Laboratories). The wavelengths for excitation andemission were 450-490 nm and 560-580 nm, respectively. The fluorescencesignal was measured at a starting temperature of 25° C. and increased to95° C. in 0.5° C./min increments. Data was analyzed and melting curvesintegrated using the Bio-Rad CFX Maestro software (v1.1). The Tm wasdefined as the local minimum taken from the derivative of the meltingcurve. The Tm of the rat antibody 2166 was 66.5° C.

A binding assay was completed to confirm binding of the antibodies toNGF (R&D Systems, 256-GF-100/CF). In addition, the specificity of theantibodies was determined by testing the binding of the antibodies toNT-3 and BDNF which are closely related proteins. Unique antibodysequences were confirmed to bind the screening target using amultiplexed bead assay on a high throughput flow cytometer. Differentoptically encoded beads were conjugated to either human NGF (R&DSystems, 256-GF-100/CF), NT-3 (R&D Systems, 267-N3-025/CF) or BDNF (R&DSystems, 248-BDB-050/CF). Purified antibodies were incubated with themultiplexed beads at different antibody concentrations for 30 minutes atroom temperature. Beads were washed and binding was detected by using afluorescently labelled secondary antibody. Fluorescence was measuredusing high throughput plate-based flow cytometry on an Intellicyt® iQueScreener Plus.

Median fluorescence intensity of each antibody was normalized over themedian fluorescence intensity of the appropriate isotype control forindividual bead types. Antibody values greater than 10-fold over isotypewere considered as binders.

Antibody 2166 bound to NGF greater than 59-fold higher than backgroundlevels and the binding of this antibody to NT-3 and BDNF was atbackground levels.

A functional assay with TF-1 cells was used to determine if the bindingof the 2166 antibody to canine NGF blocks the ability of canine NGF toinduce signaling with human TrkA which is the high affinity receptor forNGF (Chevalier et al., 1994. Blood, 83:1479). For these studies, canineNGF (Genbank NP_001181879.1) was used for the NGF source. Canine NGFwith a strep-tag (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys) at the C-terminus wasstably expressed in Dmel-2 cells and purified using StrepTactinXTchromatography followed by a polishing step with Superdex 200 16/600chromatography. The proliferation of TF-1 cells can be stimulated bydifferent growth factors such as GM-CSF and NGF. TF-1 cells(ATCC-CRL2003) were cultured in RPMI-1640 media containing 10% fetalbovine serum, 100 U/mL Penicillin, 100 μg/ml Streptomycin and 2 ng/mLrecombinant human GM-CSF. Cells were maintained between 3×10⁴ and 5×10⁵viable cells/mL and passaged every 48 hours. Each condition was run intriplicate wells. Cells were collected and counted. Cells wereresuspended in media without GM-CSF at 1.75×10⁵ cells/ml and incubatedin a flask in a humidified 37° C., 5% CO2 incubator for 4 hours. Duringthe incubation, NGF/antibody mixtures were prepared in media as 2× mediasolutions in full media without GM-CSF and with 10 ng/mL canine NGF.Antibodies were added to the appropriate 2× media solutions and theNGF/antibody solutions were incubated for at least 1 hour at roomtemperature before being added to the cells. Cells were then collectedand resuspended in appropriate media volume to achieve a 0.5×10⁶cells/ml suspension in media without GM-CSF. 50 μl of the cellsuspension was added per well in a 96-well plate, to which 50 μl of the2×NGF/antibody media was added per well to the cell plate. Cells wereincubated in a humidified 37° C., 5% CO2 incubator for 48 hours, then 20μl of Aqueous One solution Reagent (Promega) was added per well. Cellswere incubated for further 4 hours in a humidified 37° C., 5% CO2incubator and then absorbance was read at 490 nm on a BioTekSynergy/neO2. Data was analyzed by subtracting the blank well from allmeasured values. Percent inhibition was calculated using the followingformula: % inhibition=100×[1−(X−MIN)/(MAX−MIN)], where X=signal at agiven concentration, MAX=0% inhibition=Canine NGF only and MIN=100%inhibition=No NGF control. The average of the triplicates for eachcondition was calculated. The proliferation data for rat antibody 2166and the isotype rat antibody control are shown in FIG. 3 . The datademonstrates that the rat antibody 2166 effectively blocks NGF frombinding to TrkA.

The VH domain of antibody 2166 was fused with the canine IgGB constantdomains (Tang et al. 2001. Vet. Immunol. Immunopathol. 80:259) and theV_(L) domain of antibody 2166 was fused to the canine kappa constantdomain to generate a canine chimeric antibody (FIG. 4 ). Two residuechanges (AA) were made in the Fc (underlined and in bold font) toeliminate effector activity and these changes are analogous to the“LALA” mutation described for human IgG1 Fc (Tamm & Schmidt, 1997. Int.Rev. Immunol. 16:57). These two constructs were subcloned into pcDNA3.4(ThermoFisher Scientific) and co-transfected with the Expi293 system(ThermoFisher Scientific) and purified with HiTrap Protein A HPchromatography. The purity of the antibody as measured by SDS/PAGEwas >95% and by SEC (size exclusion chromatography) the antibody was 98%monomeric.

The affinity of the canine 2166 chimeric antibody for canine NGF wasmeasured by SPR (surface plasmon resonance). For these studies, canineNGF (Genbank NP_001181879.1) was generated by fusing the C-terminus withthe Flag tag (DYKDDDDK), expressing the canine NGF construct withbaculovirus technology and then purifying the NGF with Anti-DYKDDDDK G1affinity chromatography. The binding kinetics of canine 2166 chimericantibody to canine NGF was measured with a Biacore T200 instrument. Theformat of the assay was to capture the Fc of 2166 antibody onto aprotein A sensor chip and use canine NGF as the analyte. The runningbuffer was HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA,0.005% tween 20) and the instrument temperature was set at 25° C. Theflow rate was 40 μl/min and the five analyte concentrations tested induplicate ranged from 0.78 nM to 12.5 nM. The binding signals werecorrected for the blank and the resulting sensorgram (FIG. 5 ) was usedto determine the rate constants (ka and ka) and binding affinity (K_(D))using a one-to-one binding model with the BIAEVAL software. Under thesebinding conditions, the binding kinetics for canine 2166 chimericantibody were ka (1/Ms)=1.2E+7, ka (1/s)=3.5E-6, and K_(D)=3E-13. Themeasured affinity (K_(D)) of this antibody exceeds the sensitivity ofthe Biacore instrument but from these data it is estimated to be atleast 50 pM.

The ability of canine 2166 chimeric antibody to block canine NGF frombinding to the canine NGF receptors (TrkA and p75) was measured in anSPR assay on a Biacore T200. The format of the assay was to capture theNGF receptor on a sensor chip and flow over either canine NGF only, NGFmixed with canine 2166 chimeric antibody, or canine 2166 chimericantibody only.

The NGF receptors used in the assay consist of the extracellular domainsof canine p75 (XP_038340439.1) and canine TrkA (XP_038398906.1) fused tothe human IgG1 Fc (UniProtKB P01857) with a 2×Gly-Gly-Gly-Ser linkerbetween the receptor and the Fc. The fusion proteins were expressed inCHO cells and purified with protein A chromatography. For this assay,the proteins p75-Fc and Trk-Fc were captured onto a human anti-Fc sensorchip.

NGF only, canine 2166 chimeric antibody only and canine 2166 chimericantibody-NGF mixture (at a 2:1 molar ratio) were the analytes. The sevenconcentrations of NGF in NGF only condition ranged from 0.78 nM to 50nM. The concentrations of canine NGF in the canine 2166 chimericantibody-NGF mixture was 50 nM, 25 nM and 12.5 nM. Lastly, the fourconcentrations of canine 2166 chimeric antibody alone condition rangedfrom 12.5 nM to 100 nM. The instrument temperature and flow rate wereset at 25° C. and 40 μL/min, respectively.

The binding signals were corrected for the reference, and the resultingsensorgrams were used to determine the rate constants (ka and ka) andbinding affinity (K_(D)) using a one-to-one binding model with theBIAEVAL software. The sensorgrams (FIGS. 5, 6, 7, 8 ) represent the NGFonly condition and the canine 2166 chimeric antibody-NGF mixture forboth the p75-Fc and TrkA-Fc receptors. Under these binding conditions,the binding kinetics for canine NGF only to canine p75 in a dimer formatwere k_(a) (1/Ms)=1.2E+10, k_(d) (1/s)=45, and K_(D)=3.7E-9 (FIG. 6 ).The binding kinetics for canine NGF only to canine TrkA in a dimerformat were k_(a) (1/Ms)=1.3E+7, k_(d) (1/s)=1.7E-4, and K_(D)=1.3E-11(FIG. 7 ). No binding to the canine NGF receptors was observed with thecanine 2166 chimeric antibody only condition (data not shown). Asevidenced by the sensorgrams (FIGS. 8 and 9 ), canine 2166 chimericantibody effectively blocks canine NGF binding to canine TrkA and p75.

Example 2

Caninization of Rat 2166 Antibody

A canine antibody database was generated by performing NGS (nextgeneration sequencing) on canine PBMCs (peripheral blood mononuclearcells). This database contains the sequences from 5.0×10⁶ V_(H) domains,3.7×10⁶ V_(κ) domains and 2.6×10⁶ V_(L) domains. The HCDR 1, 2 and LCDR1, 2, 3 sequences from the 2166 parental antibody were used in analgorithm to identify the closest canine CDR sequences and their linkedframework sequences in the canine antibody database. These linkedframework sequences were included in the scFv phage display libraryalong with the closest framework germline sequences and the linkedframework sequences with 1 to 3 residues reverted back to the closestgermline. A proprietary algorithm was used to identify a set of CDRsequences that are similar to the original 2166 CDRs and closer inidentity to the germline and expressed CDR sequences. These CDRs andframework sequences were used to generate a scFv antibody phage displaylibrary with a theoretical complexity of 3×10¹². Antibody phageselections were completed with canine NGF for four rounds and with eachround the stringency was increased by reducing the antigen concentrationand increasing the number of washes. Specifically, 96-multi well plateswere coated with 200 pmol of NGF for the first round, 100 pmol for thesecond round and 50 pmol for the third and fourth rounds. The number ofwashes with PBS-tween 20 (0.01%) after the selection were six for thefirst round, seven for the second round, eight for the third round andnine for the fourth round. The output scFv clones from the third andfourth rounds were sequenced and unique clones were reformatted intoIgGs and screened for binding to canine NGF by SPR. The sequences andthe binding kinetics to canine NGF of the top 69 caninized clones alongwith parental clone 2166 are shown in FIGS. 1 and 2 and Table 2.

TABLE 2 Binding to canine NGF Clone ka kd KD 2166 2.38E+06 1.00E−054.20E−12 SC-42_006 2.22E+06 1.75E−04 7.88E−11 SC-42_007 1.32E+061.38E−04 1.05E−10 SC-42_008 1.96E+06 9.40E−05 4.79E−11 SC-42_0102.06E+06 8.55E−05 4.16E−11 SC-42_011 1.27E+06 7.28E−05 5.75E−11SC-42_023 4.19E+06 4.66E−04 1.11E−10 SC-42_024 3.63E+06 5.22E−041.44E−10 SC-42_025 3.90E+06 5.88E−04 1.51E−10 SC-42_026 4.26E+069.20E−04 2.16E−10 SC-42_027 3.43E+06 6.51E−04 1.90E−10 SC-42_0283.19E+06 4.96E−04 1.56E−10 SC-42_029 4.09E+06 5.44E−04 1.33E−10SC-42_030 4.10E+06 6.34E−04 1.55E−10 SC-42_031 4.23E+06 6.46E−041.53E−10 SC-42_032 4.61E+06 2.64E−04 5.73E−11 SC-42_033 3.73E+065.81E−04 1.56E−10 SC-42_034 2.81E+06 1.51E−03 5.39E−10 SC-42_0353.02E+06 6.24E−04 2.06E−10 SC-42_036 3.40E+06 8.77E−04 2.58E−10SC-42_037 3.25E+06 4.82E−04 1.49E−10 SC-42_038 3.21E+06 5.30E−041.65E−10 SC-42_040 4.82E+06 7.09E−04 1.47E−10 SC-42_041 3.42E+065.55E−04 1.62E−10 SC-42_042 3.51E+06 9.76E−04 2.78E−10 SC-42_0432.79E+06 7.13E−04 2.55E−10 SC-42_044 3.12E+06 4.53E−04 1.45E−10SC-42_045 3.34E+06 4.12E−04 1.23E−10 SC-42_046 3.45E+06 4.89E−041.42E−10 SC-42_047 3.55E+06 4.41E−04 1.24E−10 SC-42_048 4.25E+064.89E−04 1.15E−10 SC-42_049 3.80E+06 5.49E−04 1.44E−10 SC-42_0503.49E+06 5.02E−04 1.44E−10 SC-42_051 3.27E+06 6.09E−04 1.86E−10SC-42_052 3.35E+06 4.13E−04 1.23E−10 SC-42_053 3.41E+06 4.71E−041.38E−10 SC-42_054 3.32E+06 5.91E−04 1.78E−10 SC-42_055 4.23E+067.73E−04 1.83E−10 SC-42_057 3.73E+06 5.53E−04 1.48E−10 SC-42_0583.31E+06 5.88E−04 1.78E−10 SC-42_059 3.20E+06 6.38E−04 2.00E−10SC-42_060 3.87E+06 5.85E−04 1.51E−10 SC-42_061 3.16E+06 4.94E−041.56E−10 SC-42_062 3.27E+06 6.36E−04 1.95E−10 SC-42_063 3.57E+068.52E−04 2.39E−10 SC-42_064 2.43E+06 6.51E−04 2.68E−10 SC-42_0653.47E+06 5.95E−04 1.71E−10 SC-42_066 3.16E+06 5.21E−04 1.65E−10SC-42_067 3.57E+06 7.02E−04 1.97E−10 SC-42_068 3.59E+06 4.87E−041.36E−10 SC-42_069 2.39E+06 4.67E−04 1.96E−10 SC-42_070 4.70E+065.43E−04 1.15E−10 SC-42_071 1.45E+06 6.89E−04 4.74E−10 SC-42_0723.98E+06 7.86E−04 1.98E−10 SC-42_073 5.83E+06 5.46E−04 9.37E−11SC-42_075 4.54E+06 1.55E−03 3.42E−10 SC-42_077 3.35E+06 2.91E−048.71E−11 SC-42_079 3.53E+06 8.49E−04 2.40E−10 SC-42_080 3.31E+064.46E−04 1.35E−10 SC-42_081 3.41E+06 2.17E−03 6.35E−10 SC-42_0823.45E+06 2.52E−04 7.29E−11 SC-42_083 4.17E+06 6.23E−04 1.49E−10SC-42_084 3.60E+06 4.73E−04 1.32E−10 SC-42_085 4.28E+06 8.34E−041.95E−10 SC-42_088 3.39E+06 1.06E−03 3.11E−10 SC-42_089 4.26E+067.74E−04 1.82E−10 SC-42_090 1.91E+06 1.52E−04 7.99E−11 SC-42_0911.00E+04 1.00E−04 1.00E−08 SC-42_101 3.41E+06 3.80E−04 1.11E−10SC-42_102 2.91E+06 1.07E−03 3.67E−10

Sensorgrams for all 69 clones are in FIG. 10 . The SPR was completed byamine coupling the antibody (˜5 μg/ml) to the HC30M sensor chip byEDC/NHS activation, followed by ethanolamine HCL quenching. The canineNGF (Genbank NP_001181879.1) used as the analyte for the SPR analyseswas the same preparation described in Example 1 for the canine NGFtagged at the C-terminus with the Flag tag (DYKDDDDK).

The ability of caninized SC42_101 antibody to block canine NGF frombinding to the canine NGF receptors (TrkA and p75) was measured in anSPR assay on a Biacore T200. The format of the assay was to capture theNGF receptor on a sensor chip and flow over either canine NGF only, NGFmixed with caninized SC42_101 antibody, or caninized SC42_101 antibodyonly. The receptor blocking methods are identical to those described forcanine 2166 chimeric antibody in Example 1. The sensorgrams (FIGS. 11,12, 13, 14 ) represent the NGF only condition and the caninized SC42_101antibody-NGF mixture for both the p75-Fc and TrkA-Fc receptors. Underthese binding conditions, the binding kinetics for canine NGF only tocanine p75 in a dimer format were k_(a) (1/Ms)=3.8E+7, k_(d) (1/s)=0.1,and K_(D)=2.7E-9 (FIG. 11 ). The binding kinetics for canine NGF only tocanine TrkA in a dimer format were k_(a) (1/Ms)=2.4E+7, k_(d)(1/s)=1.6E-4, and K_(D)=6.6E-12 (FIG. 12 ). No binding to the canine NGFreceptors was observed with the caninized SC42_101 antibody onlycondition (data not shown). As evidenced by the sensorgrams (FIGS. 13and 14 ), caninized SC42_101 antibody effectively blocks canine NGFbinding to canine TrkA and p75.

Example 3

Felinization of Rat 2166 Antibody

A feline antibody database was generated by performing NGS (nextgeneration sequencing) on feline PBMCs (peripheral blood mononuclearcells). This database contains the sequences from 7.5×10⁶ V_(H) domains,1.3×10⁶ V_(κ) domains and 3.8×10⁶ V_(L) domains. The HCDR 1, 2 and LCDR1, 2, 3 sequences from the 2166 parental antibody were used in analgorithm to identify the closest feline CDR sequences and their linkedframework sequences in the feline antibody database. These linkedframework sequences were included in the scFv phage display libraryalong with the closest framework germline sequences and the linkedframework sequences with 1 to 3 residues reverted back to the closestgermline. A proprietary algorithm was used to identify a set of CDRsequences that are similar to the original 2166 CDRs and closer inidentity to the germline and expressed CDR sequences. These CDRs andframework sequences were used to generate a scFv antibody phage displaylibrary with a theoretical complexity of 3×10¹². The processed form offeline NGF (XP_004001166.1) is identical to the processed form of canineNGF (NP_001181879.1) so for the felininization studies, canine NGFtagged at the C-terminus with the Flag tag (DYKDDDDK) described inExample 1 was used.

Antibody phage selections were completed with NGF for four rounds andwith each round the stringency was increased by reducing the antigenconcentration and increasing the number of washes. Specifically,96-multi well plates were coated with 200 pmol of NGF for the firstround, 100 pmol for the second round and 50 pmol for the third andfourth rounds. The number of washes with PBS-tween 20 (0.01%) after theselection were six for the first round, seven for the second round,eight for the third round and nine for the fourth round. The output scFvclones from the third and fourth rounds were sequenced and unique cloneswere reformatted into IgGs and screened for binding to NGF by SPR. Thevariable domains of clone 101 are shown in FIG. 15 with the CDR regionsunderlined. The affinity of felinized clone 101 for NGF was determinedby SPR. The format of this assay was to immobilize goat anti-cat IgG (30μg/ml) on a series S CM5 biosensor using EDC/NHS and quenching theremaining sites with ethanolamine. Captured felinized clone 101 (1μg/ml) onto the goat anti-cat IgG sensor chip. Using single cyclekinetics, five concentrations of NGF were captured. The binding kineticsfor felinized clone 101 for NGF were k_(a) (1/Ms)=3.8E+5, k_(d)(1/s)=3E-3, and K_(D)=7.8E-9.

Example 4

Affinity Maturation of Felinized Clone 101 Using Site-SpecificMutagenesis of the CDRs.

In the first affinity maturation approach, the heavy variable domain andCH1 domain of feline clone 101 (Table 3) were subcloned in the GenScriptFASEBA plasmid. The construct included at the C-terminus of the heavychain (VH-CH1) a single-domain antibody against serum albumin (SASA) tag(see, e.g. US 2013/0129727A1) which has low pM affinity for albumin, andfurther downstream a His-tag for purification. The light chain variabledomain was subcloned with feline Cκ (Table 3) into a proprietary E. coliexpression vector. Both the heavy chain and light chain had the PelB(pectate lyase B) signal peptide at the N-terminus to facilitatesecretion of the Fab when expressed in TG1 E. coli. The expression ofthe variable domains was regulated by the Lac promoter.

TABLE 3 Sequences clone 101 DVQLVESGGD LVKPGGSLRL TCVASGLSLT VH-CH1SSSMSWVRQA PGKGLQWVST IYSNGGTYYT (IgG1) DSVKGRFTIS KDNAENTLYL QMNNLKTEDTATYYCASIYY YDADYLHWYF DFWGQGALVT VSSASTTAPS VFPLAPSCGT TSGATVALACLVLGYFPEPV TVSWNSGALT SGVHTFPAVL QASGLYSLSS MVTVPSSRWL EIQMTQSPTSLSASVGDRVT ITCRASEGIS NNLSWYQQTP GKAPKLLIYA SDTFTCNVAH PPSNTKVDKT Vclone 1 TSNLHSGVPS RFSGSGSGTD FTLTISSLQP VL-Cκ1EDFATYYCQQ GYKWPLTFGG GTKLEITRSD AQPSVFLFQP SLDELHTGSA SIVCILNDFYPKEVNVKWKV DGVVQNKGIQ ESTTEQNSKD STYSLSSTLT MSSTEYQSHE KFSCEVTHKSLASTLVKSEN RSECQRE

A variant library was generated for each CDR position in the heavy andlight chains using the GenScript proprietary Precision Mutant Library(PML) which utilizes semiconductor-based oligonucleotide synthesistechnology. In generating the mutants, the CDRs were defined using acombination of Kabat and IMGT methodology and the residues selected foreach CDR are shown below in Table 4. The residue numbers for the CDRsare shown in parentheses.

TABLE 4 CDR sequences and numbering VH VL CDR1 GLSLTSSSMS CDR1RASEGISNNLS (26-35) (24-34) CDR2 TIYSNGGTYYTDSVKG CDR2 ATSNLHS (50-65)(50-56) CDR3 ASIYYYDADYLHWYFDF CDR3 QQGYKWPLT  (96-112) (89-97)

The quality of the libraries was verified using NGS (Next GenerationSequencing). Forty-four PML clones were selected from each library forexpression in E. coli in 96 deep-well plates by inoculating into 2YTmedium and inducing with 0.2 mM IPTG overnight at room temperature. TheFab secreted in the medium was analyzed for binding activity bycompleting an ELISA. In this ELISA, plates were coated with 10 μg/ml ofBSA overnight at 4° C., washed 3× with 0.1% tween 20 in PBS, pH 7.4(PBST), blocked non-specific interactions with 3% non-fat dry milk inPBS (phosphate-buffered saline, pH 7.4) at 37° C. for 1 hour, washed 3×with PBST, added crude Fab supernatant (diluted 1:1 with PBST) incubatedat 37° C. for 1 hour, washed 3× with PBST, added 0.15 μg/ml of NGFincubated at 37° C. for 1 hour, washed 3× with PBST, added horseradishperoxidase (HRP) conjugated anti-Flag tag antibody (Flag tag present onNGF) incubated at room temperature for 45 minutes, washed 3× with PBSTand detected the HRP conjugate by incubating with TMB substrate for 10minutes at room temperature and measured absorbance at 450 nm. The top100 clones with an apparent increase in affinity as measured by ELISAwere sequenced to detect the variant in the CDR and 57 unique cloneswere identified. Mutations from clone 101 for each of the 57 clones aretallied in Table 5.

TABLE 5 Individual mutations at each position compared to clone 101CDR1H CDR2H CDR3H 28 30 31 35 52 53 55 58 60 62 64 97 99 101 104 112 101S T S S Y S G Y T S K S Y Y D F H N H V W P R D H D E Q F H K E R Y D DM E P S N N H E K Q N H T CDR1L CDR2L 24 30 31 34 53 54 55 56 101 R S NS N L H S F A Q G I H M T Q A V I D V K L E L H N P M Y L

Binding of the 57 unique clones were confirmed by an off-rate screeningassay in an SPR assay performed on a Biacore T200. For the SPR analyses,bovine serum albumin (BSA) was immobilized to CM5 sensor chip. Thesensor chip surface was activated with 50 mmol/L H-Hydroxysuccinimideand 200 mmol/L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride for 420 s. Afterwards, BSA diluted in 10 mM sodiumacetate, pH 4.5 was injected. After the amine coupling reaction, theremaining active coupling sites on the chip surface were blocked with 1mM ethanolamine hydrochloride. The selected Fab-SASA variants inconditioned medium were captured on the BSA-coated chips. The runningbuffer was HBS-EP (10 mM HEPES 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20,pH 7.4). After equilibration, antigen was injected for 120 seconds(association phase) followed by the injection of running buffer for 420sec (dissociation phase). The off-rates of the Fab-SASA clones wereobtained from fitting the experimental data locally to a 1:1 interactionmodel using the Biacore T200 evaluation software. The Fab variants wereranked by their dissociation rate constants (off-rates, kd) shown inTable 6.

TABLE 6 Rank ordering of the Fab variants based on dissociationconstants Ratio Sequence Rmax Chi² WT-kd/ Analysis ka (1/Ms) kd (1/s) KD(M) (RU) (RU²) clone-kd G55R 1.20E+05 1.47E−04 1.23E−09 19.7 0.006346.41 S30A 2.77E+05 2.92E−04 1.05E−09 152.2 0.618 3.23 H55M 3.33E+053.12E−04 9.35E−10 137.7 0.712 3.02 S30Q 2.59E+05 3.24E−04 1.25E−09 132.50.462 2.91 D104K 6.09E+04 3.32E−04 5.45E−09 64.6 0.0316 2.84 S35V3.09E+05 3.60E−04 1.17E−09 106.1 0.414 2.62 N53I 2.76E+05 3.89E−041.41E−09 118 0.347 2.42 F112E 2.84E+05 3.99E−04 1.41E−09 147.2 0.4882.36 S97Q 2.97E+05 4.03E−04 1.36E−09 79.5 0.287 2.34 N53V 2.88E+054.07E−04 1.42E−09 108.7 0.375 2.32 H55L 2.64E+05 4.18E−04 1.58E−09 91.80.494 2.26 G55Y 2.29E+05 4.19E−04 1.83E−09 17.7 0.0131 2.25 D104E2.37E+05 4.26E−04 1.80E−09 66.4 0.126 2.21 H55I 3.34E+05 4.36E−041.31E−09 100.7 0.67 2.16 D104N 2.39E+05 4.46E−04 1.87E−09 52.8 0.07742.11 S97M 2.66E+05 4.66E−04 1.75E−09 64.3 0.149 2.02 Y99F 3.21E+054.69E−04 1.46E−09 100.8 0.439 2.01 K64E 1.96E+05 4.76E−04 2.42E−09 50.20.164 1.98 D104Q 2.20E+05 4.78E−04 2.17E−09 49.6 0.056 1.97 N31Q3.27E+05 4.80E−04 1.47E−09 80.2 0.343 1.96 S97N 3.73E+05 4.81E−041.29E−09 69.6 0.317 1.96 S30V 2.87E+05 4.83E−04 1.68E−09 59.3 0.22 1.95Y101H 3.07E+05 4.96E−04 1.62E−09 61.8 0.194 1.90 S30L 2.70E+05 5.07E−041.87E−09 92.6 0.914 1.86 S97K 3.31E+05 5.11E−04 1.54E−09 62.3 0.226 1.85S34G 4.56E+05 5.14E−04 1.13E−09 65.8 0.571 1.83 S97H 2.97E+05 5.33E−041.79E−09 72 0.198 1.77 F112P 3.98E+05 5.52E−04 1.39E−09 48 0.164 1.71K64D 3.40E+05 5.54E−04 1.63E−09 48.1 0.189 1.70 S62D 3.01E+05 5.60E−041.86E−09 103.3 0.405 1.68 L54H 3.40E+05 5.60E−04 1.65E−09 75.6 0.3571.68 T60H 3.12E+05 5.61E−04 1.80E−09 77.5 0.316 1.68 Y58D 3.81E+055.62E−04 1.47E−09 108.4 0.571 1.68 T60D 2.94E+05 5.67E−04 1.93E−09 90.60.299 1.66 T60S 3.25E+05 5.73E−04 1.76E−09 80.6 0.33 1.65 N53K 2.92E+055.96E−04 2.04E−09 60.5 0.172 1.58 S31H 3.10E+05 6.12E−04 1.98E−09 66.60.291 1.54 N53H 3.22E+05 6.27E−04 1.95E−09 69.4 0.177 1.50 F112H3.28E+05 6.39E−04 1.95E−09 81.8 0.407 1.48 F112N 3.47E+05 6.43E−041.85E−09 72 0.316 1.47 N53M 3.46E+05 6.48E−04 1.87E−09 53.7 0.312 1.46Y52W 4.34E+05 6.51E−04 1.50E−09 41.3 0.258 1.45 R24F 2.87E+05 6.55E−042.28E−09 89.3 0.273 1.44 N53L 3.21E+05 6.67E−04 2.08E−09 60 0.188 1.41S30P 2.62E+05 6.70E−04 2.56E−09 75.4 0.211 1.41 T60E 2.87E+05 6.83E−042.38E−09 77.7 0.289 1.38 S53P 3.23E+05 6.95E−04 2.15E−09 56.6 0.183 1.36T30N 3.33E+05 7.04E−04 2.12E−09 71.2 0.287 1.34 F112E 2.94E+05 7.08E−042.41E−09 64.9 0.221 1.33 S56T 3.58E+05 7.14E−04 1.99E−09 65.5 0.414 1.32S97T 3.80E+05 7.16E−04 1.88E−09 51.1 0.207 1.32 T30R 3.57E+05 7.30E−042.05E−09 61.2 0.261 1.29 S56D 3.72E+05 7.55E−04 2.03E−09 49.1 0.224 1.25S30Y 3.55E+05 7.82E−04 2.20E−09 60.5 0.4 1.21 S34A 4.50E+05 7.83E−041.74E−09 50.4 0.361 1.20 S28H 3.15E+05 8.02E−04 2.55E−09 79.8 0.244 1.18S56E 3.70E+05 8.09E−04 2.19E−09 54.6 0.249 1.17 S56N 3.41E+05 9.03E−042.64E−09 50.6 0.178 1.04 Wild-Type 3.59E+05 9.39E−04 2.62E−09 59.1 0.2411.00 Wild-Type 3.55E+05 9.43E−04 2.655E−09  1.00 Wild-Type 3.51E+059.47E−04 2.69E−09 60.1 0.235 1.00

Fab variants G55R, S30A, S30Q, S35V, N53I, F112E, S97Q, and N53V wereselected for combinatorial library construction (Table 7).

TABLE 7 Selected Fab variants for combinatorial library VH VL Position35 55 97 112 30 53 Wild-Type S G S F S N Variant V R Q E A I Q V

The combinatorial library was constructed in the same Fab-SASA vectordescribed above. The theoretical diversity of the combinatorial libraryis 2×2×2×2×3×3=144 and the size of the constructed library was 5.6×10⁷CFU (colony forming units). The library in-frame rate and diversity wereevaluated by DNA sequencing and the results are shown in the tablesbelow.

TABLE 8 Percentage of in-frame Fab-SASA construct in combinatoriallibrary Clones for Sequences with sequencing stop codons In-frame rateUnique clones VH 47 3 44/47 = 93.6% 12 VL 47 2 45/47 = 95.7% 9

TABLE 9 Diversity of heavy chain variants in combinatorial libraryNumber of Chain clones S35V G55R S97Q F112E VH 44 S(30), V(14) G(10),R(34) S(36), Q(8) F(33), E(11)

TABLE 10 Diversity of light chain variants in combinatorial libraryNumber of Chain clones S30A/Q N53I/V VL 43 S(16), A(21), Q(8) N(13),I(17), V(15)

From the combinatorial library, 184 clones were randomly selected andthe binding by the NGF ELISA was completed. The NGF ELISA was the samemethod as described above. The top 20 clones in ELISA binding weresequenced and tested in the SPR off-rate assay. Binding results areshown by amino acid combination in Table 11. Table 12 indicates variabledomain sequence IDs for the top 20 clones. The pairings of V_(H) andV_(L) indicate substantial compatibility of the V_(H) and V_(L)mutations and interchangeability of the V_(H) and V_(L) domains.

TABLE 11 Affinity matured Fab variants from the combinatorial libraryRatio Ratio VH VL (kd) (KD) Antibody 35 55 97 112 30 53 ka kd KD WT/ WT/Rmax ID S G S F S N (1/Ms) (1/s) (M) clones clones (RU) AHF17591 V R S EA I 2.68E+05 1.00E−06 3.73E−12 1685.00 925.94 19.3 AHF17598 V R S E A I2.94E+05 5.25E−05 1.79E−10 32.10 19.30 25.9 AHF17592 V R S F S V1.92E+05 1.46E−05 7.61E−11 115.41 45.40 28.5 AHF17600 V R S F S V1.77E+05 5.81E−05 3.29E−10 29.00 10.50 26.7 AHF17593 V R Q F A V1.63E+05 1.00E−06 6.13E−12 1685.00 563.17 24.4 AHF17594 V G S F A V3.53E+05 3.28E−04 9.31E−10 5.14 3.71 86.1 AHF17595 V R S E Q N 1.78E+051.00E−06 5.62E−12 1685.00 614.99 41.6 AHF17596 V R S F A V 2.27E+051.82E−05 8.02E−11 92.58 43.08 31.5 AHF17608 V R S F A V 2.28E+051.00E−06 4.39E−12 1685.00 787.74 28.8 AHF17599 V R S F A V 2.12E+051.00E−06 4.72E−12 1685.00 732.46 34.9 AHF17597 V R S F Q I 1.99E+051.08E−05 5.44E−11 156.02 63.51 37.6 AHF17601 V R Q E A N 2.91E+051.00E−06 3.44E−12 1685.00 1005.41 42.6 AHF17602 V R S F A N 2.10E+051.00E−06 4.76E−12 1685.00 725.55 27.9 AHF17606 V R S F A N 1.87E+052.01E−05 1.07E−10 83.83 32.29 46.3 AHF17610 V R S F A N 1.82E+051.00E−06 5.49E−12 1685.00 628.81 35 AHF17610 V R S F A N 1.82E+051.00E−06 5.49E−12 1685.00 628.81 35 AHF17603 V G S E A I 3.68E+052.45E−04 6.64E−10 6.88 5.20 108.7 AHF17604 V R Q F Q I 1.45E+05 4.66E−053.21E−10 36.16 10.76 27.9 AHF17605 V G Q F A I 2.53E+05 2.07E−048.20E−10 8.14 4.21 94.2 AHF17607 V R S E S I 2.26E+05 1.00E−06 4.42E−121685.00 780.83 36.9 AHF17609 V R S F A I 2.28E+05 1.00E−06 4.39E−121685.00 787.74 35.9 Blank 1.92E+04 NA NA NA NA NA Blank 1.45E+04 NA NANA NA NA Wild-type 5.69E+05 1.79E−03 3.15E−09 0.94 0.00 28.7 Wild-type4.19E+05 1.58E−03 3.78E−09 1.07 0.00 30.2 Wild-type 4.94E+05 1.69E−033.47E−09 1.00 0.00 29.45

TABLE 12 VH and VL variable domain sequences of affinity matured Fabvariants VH VL Antibody 35 55 97 112 30 53 ID SEQ ID NO: S G S F SEQ IDNO: S N AHF17591 184 V R S E 198 A I AHF17598 184 V R S E 198 A IAHF17592 188 V R S F 192 S V AHF17600 188 V R S F 192 S V AHF17593 185 VR Q F 193 A V AHF17594 186 V G S F 193 A V AHF17595 184 V R S E 194 Q NAHF17596 188 V R S F 193 A V AHF17608 188 V R S F 193 A V AHF17599 188 VR S F 193 A V AHF17597 188 V R S F 195 Q I AHF17601 187 V R Q E 196 A NAHF17602 188 V R S F 196 A N AHF17606 188 V R S F 196 A N AHF17610 188 VR S F 196 A N AHF17610 188 V R S F 196 A N AHF17603 189 V G S E 198 A IAHF17604 187 V R Q F 195 Q I AHF17605 190 V G Q F 198 A I AHF17607 184 VR S E 197 S I AHF17609 188 V R S F 198 A I

Example 5

Affinity Maturation of Felinized Clone 101 by scFv Phage Display

In the second affinity maturation approach, a scFv phage display librarywas constructed containing the frameworks of felinized clone 101 and thefollowing sequences for the heavy and light CDR sequences shown in Table13.

TABLE 13 CDRs of felinized clone 101 and variants inscFv phage display combinatorial library HCDR1 HCDR2 HCDR3 LCDR1 LCDR2LCDR3 GLSLTSSS IYSNGGT ASIYYYDADYLHWYFDF EGISNN ATS QQGYKWPLT ------H--W----- -N--------------- Q----- --E ---W----- -----TN- ---Q----E--------------- N----- -S- ---H----- --E---N- --A-----Q--------------- K----- --N ---F----- A------- ---E----D--------------- -A---- --D ----R---- L------- ---R----K--------------- --L--- --Q ----E---- N------- ---S-----L-------------- --V--- --K ----S---- --E----- LW--------T------------- ---G-- -A- ----Q---- --G----- -W-S------W------------- ---D-- -Q- -----F--- --A----- -W-E------F------------- ----S- -D- -----I--- --D----- -W-D-------H------------ ----K- -Y- ------E-- --T----- ----F------------ ----Y--V- -------M- ---M---- ------E---------- -----S -L- ---S----- ---V----------S---------- -----G ---T----- ----A--- ------V---------- -----Q---H----- ----S--- -------E--------- -----E ---G----- ----V-----------H-------- -----K ---L----- ----N--- -A--------------- -----D---V----- ----M--- -Y--------------- -----T ---R----- -----TH--T--------------- -----L ---D----- -----H-- -V--------------- -----A---K----- -----G-- -L--------------- -----H --Y-ST-W- -----E---P--------------- -----F -----R-- -H--------------- -----R -----K---R--------------- ------I- -I--------------- ------T- -G---------------------D- --Y-------------- ------N- S---------------- ------Q-T---------------- ------A- D---------------- Q------- N----------------------Y- E---------------- -------A Q---------------- -----A--K---------------- --Y----- --Y-------------M --M----- --W---------------D-F------------- -N-W------------- -Q--F------------ ------E-E---------------SY-------- --------Y-------- -----------Y----- ------------F-------------F------- -------------L--- -------------W--- -------------I---

The library diversity for the heavy chain was 37 (HCDR1)×11 (HCDR2)×57(HCDR3)=23,199 and for the light chain was 24 (LCDR1)×13 (LCDR2)×22(LCDR3)=6, 864. The library containing the combined heavy and lightchains has a diversity of 1.59×10⁸. Antibody phage selections werecompleted with NGF for five rounds and with each round the stringencywas increased by reducing the antigen concentration and increasing thenumber of washes. Specifically, 96-multi-well plates were coated with200 pmol of NGF for the first round, pmol of NGF for the second andthird rounds, 25 pmol for the fourth round and 10 pmol for the fifthround. The number of washes with PBS, pH 7.4-Tween 20 (0.01%) after theselection step was three after the first round, four after the secondround, five after the third round, six after the fourth round and sevenafter the fifth round. Isolated 760 clonal phage from each of theoutputs of the third, fourth and fifth rounds that were screened in anNGF-binding ELISA. The positive clones were sequenced and 140 uniquepositive clones were reformatted into feline IgG1a, expressed in CHOcells and purified with protein A. The SPR was completed by aminecoupling the antibody (˜5 μg/ml) to the HC30M sensor chip by EDC/NHSactivation, followed by ethanolamine HCL quenching. NGF was the analytediluted in HEPES-buffered saline with 0.01% tween 20 and 0.5 mg/ml BSA.The NGF was run at concentrations of 500 nM, 166 nM, 55 nM, 18 nM, 6.2nM, 2.0 nM, 0.68 nM, and 0.23 nM. The affinities of the top threeaffinity-matured clones are shown below in Table 14. The sequences ofvariable domains of the top three clones (SC-184_76; SC-184_102;SC-184_110) are shown in FIG. 17

TABLE 14 SPR data of top three affinity-matured feline antibodies Cloneka (1/Ms) kd (1/s) KD (M) 101 1.10E+05 2.20E−04 2.10E−09 SC-184_762.70E+05 6.90E−05 2.50E−10 SC-184_102 2.10E+05 6.30E−05 6.30E−10SC-184_110 1.60E+05 3.60E−05 3.60E−10

Example 6

SPR and NGF Receptor Blocking Data of Affinity-Matured Feline AntibodiesDirected Against Feline NGF

Affinity-matured antibodies AHF17602, SC-184_76, SC-184_102, andSC-184_110 along with the latter three clones containing the G55Rmutation (SC-184_76-Arg, SC-184_102-Arg, and SC-184_110-Arg) describedin the first affinity maturation approach were evaluated for theiraffinity to NGF using SPR with a Biacore T200 instrument. The variabledomain sequences of AHF17602, SC-184_76-Arg, SC-184_102-Arg, andSC-184_110-Arg are shown in FIG. 17 . In addition, clone 101 was alsoevaluated. Antibodies were captured using an anti-feline coupled CM5chip. NGF binding was then assessed at multiple concentrations startingat 50 nM using PBSP+ running buffer (Cytiva) with a flow rate of 30μL/min. The length of the association time was 120s and the dissociationtime was run for 600s. The chip surface was regenerated with 10 mMglycine. Reference-subtracted sensorgrams were fitted to a 1:1 bindingmodel using Biacore T100 Evaluation software. The data is shown in Table15 below and the sensorgrams in FIG. 18 .

TABLE 15 Affinity of feline NGF antibodies Clone ka (1/Ms) kd (1/s) KDRmax (RU) 101 7.84E+05 1.38E−03 1.75E−09 26.3 AHF17602 3.94E+05 6.32E−051.61E−10 61.4 SC-184_76 8.38E+05 3.96E−04 4.72E−10 61.9 SC-184_76-Arg5.85E+05 9.01E−05 1.54E−10 73.4 SC-184_102 4.71E+05 1.98E−04 4.20E−1082.1 SC-184_102-Arg 3.43E+05 4.43E−05 1.29E−10 87.8 SC-184_110 4.92E+053.80E−04 7.73E−10 43.2 SC-184_110-Arg 3.87E+05 5.48E−05 1.42E−10 53.2

For the NGF receptor blocking experiments the feline TrkA and p75 NGFreceptors were generated and used in an SPR experiment with a BiacoreT200. The extracellular domain of feline TrkA (XP_023103311) was clonedwith an AviTag (GLNDIFEAQKIEWHE) and 8×His tag at the C-terminus andexpressed in HEK293 cells. The recombinant feline TrkA protein waspurified from the conditioned medium using nickel chromatography. Theextracellular domain of feline p75 (XP_023099534) was cloned with anAviTag (GLNDIFEAQKIEWHE) and 8×His tag at the C-terminus and expressedin HEK293 cells. The recombinant feline p75 protein was purified fromthe conditioned medium using nickel chromatography. Both receptors werebiotinylated at the AviTag site using the BirA biotin protein ligasereaction kit (Avidity). Biotinylated receptors were captured on a SeriesS CAP chip and Biotin CAPture reagent (Cytiva). Antibodies were titratedin running buffer (1×PBSP+, Cytiva) and pre-incubated with 10 nM NGF(TrkA assay) or 50 nM NGF (p75 assay) at the indicated ratios. Bindingwas assessed by injecting these samples over the captured receptor for180s. The Rmax was used to calculate the inhibition percent by dividingthe Rmax of the pre-mixed samples by an average of the NGF-only Rmaxsamples that were collected throughout the assay. The ability of eachantibody to block binding of NGF to feline TrkA and p75 are shown inTable 16.

TABLE 16 Antibody blocking NGF ability to bind to the NGF receptor TrkAp75 Ab to NGF % Ab to NGF % Clone ratio Block ratio Block 101 10 to 1 151 to 1 42 101 50 to 1 76 5 to 1 87 SC-184_76-Arg 10 to 1 99 1 to 1 100SC-184_76-Arg 50 to 1 100 5 to 1 100 SC-184_110-Arg 10 to 1 100 1 to 1100 SC-184_110-Arg 50 to 1 100 5 to 1 100 AHF17602 10 to 1 99 1 to 1 100AHF17602 50 to 1 100 5 to 1 100

Example 7

Testing the Affinity Maturation Mutation G55R in the Canine CloneSC-42_101_006.

The feline clone 101, has significant CDR similarity as the canine cloneSC-42_101_006 (V_(H) domain of SC-42_101; V_(L) domain of SC-42_006).Affinity-matured feline clone AHF17602 removes a potential NGdeamidation site by mutation of the G55R and this potential deamidationsite exists in the canine clones as well. Clone SC-42_101_006 wasmutated to R55 and both the parental and the R55 variant weretransiently expressed in CHO cells and purified by Protein A. Thevariable domain sequences of are shown in FIG. 1 and FIG. 2 . Affinityto NGF was assessed using SPR with a Biacore T200 instrument. Antibodieswere captured using a Protein A Series S chip. NGF binding was thenassessed at multiple concentrations starting at 50 nM using PBSP+running buffer (Cytiva) with a flow rate of 30 μL/min. The length of theassociation time was 120s and the dissociation time was run for 600s.The chip surface was regenerated with 10 mM glycine.Reference-subtracted sensorgrams were fitted to a 1:1 binding modelusing Biacore T200 Evaluation software. The data are shown in Table 17below.

TABLE 17 Affinity of canine NGF antibodies Clone ka (1/Ms) kd (1/s) KD(M) Rmax (RU) SC-42_101_006 1.51E+06 1.74E−04 1.15E−10 46.7SC-42_101_006-Arg 1.38E+06 1.89E−04 1.39E−10 39

The invention is further described by the following numbered paragraphs:

-   -   1. A isolated protein that specifically binds to canine NGF,        which comprises an antigen binding portion that comprises:    -   (a) a heavy chain complementarity determining region 1 (VH-CDR1)        comprising the amino acid sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID        NO:146), wherein X₁ comprises A, G, or N, X₂ comprises L or M,        X₃ comprises A, D, E, or S, X₄ comprises F, I, L, M, or V, X₅        comprises N or T, X₆ comprises E, S, or T, X₇ comprises G, H, N,        S, or Q, and X₈ comprises A or S;    -   (b) a heavy chain complementarity determining region 2 (VH-CDR2)        comprising the amino acid sequence X₁X₂SNGGT (SEQ ID NO:147),        wherein X₁ comprises I or L, X₂ comprises W or Y;    -   (c) a heavy chain complementarity determining region 3 (VH-CDR3)        comprising the amino acid sequence        AX₂IX₄X₅YX₇X₈X₉YLX₁₂X₁₃YX₁₅X₁₆X₁₇ (SEQ ID NO:148), wherein X₂        comprises D, E, K, N, Q, S, or T, X₄ comprises W or Y, X₅        comprises F, H, W, or Y, X₇ comprises D or E, X₈ comprises A or        S, X₉ comprises D or Y, X₁₂ comprises H or Y, X₁₃ comprises F or        W, X₁₅ comprises F, I, L, W, or Y, X₁₆ comprises D or Q, and X₁₇        comprises F, I, L, M, W, or Y;    -   (d) a light chain complementarity determining region 1 (VL-CDR1)        comprising the amino acid sequence X₁X₂IX₄X₅X₆ (SEQ ID NO:149),        wherein X₁ comprises D, E, or K, X₂ comprises A, G, or N, X₄        comprises G, N, Q or S, X₅ comprises N or S, X₆ comprises A, G,        N, S or T;    -   (e) a light chain complementarity determining region 2 (VL-CDR2)        comprising the amino acid sequence AX₂X₃ (SEQ ID NO:150),        wherein X₂ comprises A, S, or T, X₃ comprises A, D, E, N, Q, S,        or T; and    -   (f) a light chain complementarity determining region 3 (VL-CDR3)        comprising the amino acid sequence QX₂GX₄X₅X₆PX₈T (SEQ ID        NO:151), wherein X₂ comprises H or Q, X₄ comprises F, H, W, or        Y, X₅ comprises K or Q, X₆ comprises F or W, and X₈ comprises L        or M.    -   2. The protein of paragraph 1, which comprises an antigen        binding portion that comprises:    -   (a) a heavy chain complementarity determining region 1 (VH-CDR1)        comprising the amino acid sequence X₁X₂X₃X₄TX₆X₇S (SEQ ID        NO:152), wherein X₁ comprises A or G, X₂ comprises L or M, X₃        comprises E or S, X₄ comprises F or L, X₆ comprises S or T, and        X₇ comprises H, N, or S;    -   (b) a heavy chain complementarity determining region 2 (VH-CDR2)        comprising the amino acid sequence IWSNGGT (SEQ ID NO:153);    -   (c) a heavy chain complementarity determining region 3 (VH-CDR3)        comprising the amino acid sequence AX₂IYYYX₇ADYLHXDYX₁₅DX₁₇ (SEQ        ID NO:154) comprises, wherein X₂ comprises N, Q, or S, X₇        comprises D or E, X₁₃ comprises F or W, X₁₅ comprises F, I, L,        W, or Y, and X₁₇ comprises F, I, L, or M;    -   (d) a light chain complementarity determining region 1 (VL-CDR1)        comprising the amino acid sequence X₁GIX₄NX₆ (SEQ ID NO:155),        wherein X₁ comprises D or E, X₄ comprises Q or S, X₆ comprises        G, N, S or T;    -   (e) a light chain complementarity determining region 2 (VL-CDR2)        comprising the amino acid sequence ATX₃ (SEQ ID NO:156), wherein        X₃ comprises D, E, N, Q, or S; and    -   (f) a light chain complementarity determining region 3 (VL-CDR3)        comprising the amino acid sequence QQGX₄X₅X₆PLT (SEQ ID NO:157),        wherein X₄ comprises F, H, or Y, X₅ comprises K or Q, and X₆        comprises F or W.    -   3. The protein of paragraph 1 or 2, which comprises no more than        two (2) changes per VH-CDR as compared to SEQ ID NO:137 and no        more than two (2) changes per VL-CDR as compared to SEQ ID        NO:138.    -   4. The protein of paragraph 1 or 2, which comprises no more than        one (1) changes per VH-CDR as compared to SEQ ID NO:137 and no        more than one (1) change per VL-CDR as compared to SEQ ID        NO:138.    -   5. The protein of any one of paragraphs 1 to 4, which comprises        a heavy chain framework (FR1H+FR2H+FR3H+FR4H) at least 75%, or        at least 80%, or at least 85%, or at least 90%, or at least 93%,        or at least 95% identical to SEQ ID NO:13, SEQ ID NO:31, SEQ ID        NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103,        SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ        ID NO:137, or SEQ ID NO:141.    -   6. The protein of any one of paragraphs 1 to 5, which comprises        a light chain framework (FR1L+FR2L+FR3L+FR4L) at least 75%, or        at least 80%, or at least 85%, or at least 90%, or at least 93%,        or at least 95% identical to SEQ ID NO:14, SEQ ID NO:32, SEQ ID        NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104,        SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ        ID NO:138, or SEQ ID NO:142.    -   7. The protein of any one of paragraphs 1 to 6, which comprises        a V_(H) domain comprising SEQ ID NO:13, SEQ ID NO:31, SEQ ID        NO:55, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103,        SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ        ID NO:137, or SEQ ID NO:141.    -   8. The protein of any one of paragraphs 1 to 7, which comprises        a V_(L) domain comprising SEQ ID NO:14, SEQ ID NO:32, SEQ ID        NO:56, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:104,        SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQ        ID NO:138, or SEQ ID NO:142.    -   9. An isolated nucleic acid sequence encoding an anti-NGF        antibody or antibody fragment of any one of paragraphs 1 to 8.    -   10. A vector that comprises the nucleic acid of paragraph 9.    -   11. A recombinant cell which comprises the nucleic acid of any        one of paragraphs 9 or 10.    -   12. A cell that expresses the protein of any one of paragraphs 1        therapeutically effective amount of the anti-NGF protein of any        one of paragraphs 1 to 8 or the nucleic acid of paragraphs 9 or        10.    -   13. A method of producing the anti-NGF protein of any one of        paragraphs 1 to 8, which comprises culturing the host cell of        paragraph 11 under conditions that result in production of the        anti-NGF protein.    -   14. A pharmaceutical composition comprising a therapeutically        effective amount of the anti-NGF protein of any one of        paragraphs 1 to 8.    -   15. A method of treating pain in a subject which comprises        administering to the subject a therapeutically effective amount        of the anti-NGF protein of any one of paragraphs 1 to 8.    -   16. The method of paragraph 15, wherein the pain comprises        inflammatory pain, post-operative incision pain, cancer pain,        primary or metastatic bone cancer pain, fracture pain,        osteoporotic fracture pain, pain resulting from burn, pain from        trauma, musculoskeletal pain, rheumatic pain, or osteoporosis        pain.    -   17. The method of paragraph 16, wherein the subject comprises a        canine.    -   18. The method of paragraph 16, wherein the subject comprises a        feline.    -   19. The method of paragraph 16, wherein the subject comprises a        human.    -   20. A method of detecting NGF in a sample comprising incubating        a sample comprising NGF in the presence of an anti-NGF protein        of any one paragraphs 1 to 8 and detecting the anti-NGF protein        bound to NGF in the sample.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. An antigen binding protein that specificallybinds to nerve growth factor (NGF), which comprises: (a) a heavy chaincomplementarity determining region 1 (VH-CDR1) comprising the amino acidsequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO:146), wherein X₁ comprises A, G, orN, X₂ comprises L or M, X₃ comprises A, D, E, or S, X₄ comprises F, I,L, M, or V, X₅ comprises N or T, X₆ comprises E, S, or T, X₇ comprisesG, H, N, S, or Q, and X₈ comprises A or S; (b) a heavy chaincomplementarity determining region 2 (VH-CDR2) comprising the amino acidsequence X₁X₂SNX₅GT (SEQ ID NO:147), wherein X₁ comprises I or L, X₂comprises W or Y, and X₅ comprises G or R; (c) a heavy chaincomplementarity determining region 3 (VH-CDR3) comprising the amino acidsequence AX₂₁X₄X₅YX₇X₈X₉YLX₁₂X₁₃YX₁₅X₁₆X₁₇ (SEQ ID NO:148), wherein X₂comprises D, E, K, N, Q, S, or T, X₄ comprises W or Y, X₅ comprises F,H, W, or Y, X₇ comprises D or E, X₈ comprises A or S, X₉ comprises D orY, X₁₂ comprises H or Y, X₁₃ comprises F or W, X₁₅ comprises F, I, L, W,or Y, X₁₆ comprises D or Q, and X₁₇ comprises F, I, L, M, W, or Y; (d) alight chain complementarity determining region 1 (VL-CDR1) comprisingthe amino acid sequence X₁X₂IX₄X₅X₆ (SEQ ID NO:149), wherein X₁comprises D, E, or K, X₂ comprises A, G, or N, X₄ comprises G, N, Q orS, X₅ comprises N or S, X₆ comprises A, G, N, S or T; (e) a light chaincomplementarity determining region 2 (VL-CDR2) comprising the amino acidsequence AX₂X₃ (SEQ ID NO:150), wherein X₂ comprises A, S, or T, X₃comprises A, D, E, N, Q, S, or T; and (f) a light chain complementaritydetermining region 3 (VL-CDR3) comprising the amino acid sequenceQX₂GX₄X₅X₆PX₈T (SEQ ID NO:151), wherein X₂ comprises H or Q, X₄comprises F, H, W, or Y, X₅ comprises K or Q, X₆ comprises F or W, andX₈ comprises L or M.
 2. The antigen binding protein of claim 1, whichcomprises: (a) a heavy chain complementarity determining region 1(VH-CDR1) comprising the amino acid sequence X₁X₂X₃X₄TX₆X₇S (SEQ IDNO:152), wherein X₁ comprises A or G, X₂ comprises L or M, X₃ comprisesE or S, X₄ comprises F or L, X₆ comprises S or T, and X₇ comprises H, N,or S; (b) a heavy chain complementarity determining region 2 (VH-CDR2)comprising the amino acid sequence IWSNX₅GT (SEQ ID NO:153), wherein X₅comprises G or R; (c) a heavy chain complementarity determining region 3(VH-CDR3) comprising the amino acid sequence AX₂IYYYX₇ADYLHXDYX₁₅DX₁₇(SEQ ID NO:154) comprises, wherein X₂ comprises N, Q, or S, X₇ comprisesD or E, X₁₃ comprises F or W, X₁₅ comprises F, I, L, W, or Y, and X₁₇comprises F, I, L, or M; (d) a light chain complementarity determiningregion 1 (VL-CDR1) comprising the amino acid sequence X₁GIX₄NX₆ (SEQ IDNO:155), wherein X₁ comprises D or E, X₄ comprises Q or S, X₆ comprisesG, N, S or T; (e) a light chain complementarity determining region 2(VL-CDR2) comprising the amino acid sequence ATX₃ (SEQ ID NO:156),wherein X₃ comprises D, E, N, Q, or S; and (f) a light chaincomplementarity determining region 3 (VL-CDR3) comprising the amino acidsequence QQGX₄X₅X₆PLT (SEQ ID NO:157), wherein X₄ comprises F, H, or Y,X₅ comprises K or Q, and X₆ comprises F or W.
 3. The antigen bindingprotein of claim 1, which comprises no more than two (2) substitutionsper VH-CDR as compared to SEQ ID NO:137 and no more than two (2)substitutions per VL-CDR as compared to SEQ ID NO:138.
 4. The antigenbinding protein of claim 1, which comprises no more than one (1)substitution per VH-CDR as compared to SEQ ID NO:137 and no more thanone (1) substitution per VL-CDR as compared to SEQ ID NO:138.
 5. Theantigen binding protein of claim 1, which comprises one or more VH-CDRsof any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ IDNO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ IDNO:109, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQID NO:141, or SEQ ID NO:207 and one or more VL-CDRs of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:32, SEQ ID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:70, SEQ ID NO:78, SEQ ID NO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ IDNO:122, SEQ ID NO:134, SEQ ID NO:138, or SEQ ID NO:142.
 6. The antigenbinding protein of claim 1, which comprises the VH-CDRs of any one ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:31, SEQ ID NO:55, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:103, SEQ ID NO:109, SEQ IDNO:113, SEQ ID NO:121, SEQ ID NO:133, SEQ ID NO:137, SEQ ID NO:141, orSEQ ID NO:207 and the VL-CDRs of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:32, SEQID NO:56, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ IDNO:104, SEQ ID NO:110, SEQ ID NO:114, SEQ ID NO:122, SEQ ID NO:134, SEQID NO:138, or SEQ ID NO:142.
 7. The antigen binding protein of claim 2,which comprises no more than two (2) substitutions per VH-CDR ascompared to SEQ ID NO:137 and no more than two (2) substitutions perVL-CDR as compared to SEQ ID NO:138.
 8. The antigen binding protein ofclaim 2, which comprises no more than one (1) substitution per VH-CDR ascompared to SEQ ID NO:137 and no more than one (1) substitution perVL-CDR as compared to SEQ ID NO:138.